Protein tyrosine kinases

ABSTRACT

The protein tyrosine kinase receptors, designated Rse and HPTK6, have been purified from human and/or murine cell tissues. Rse and HPTK6 have been cloned from a cDNA library of a human liver carcinoma cell line (i.e., Hep 3B) using PCR amplification. Provided herein are nucleic acid sequences encoding Rse and HPTK6 useful as diagnostics and in the recombinant preparation of Rse and HPTK6. Rse and HPTK6 are used in the preparation and purification of antibodies thereto and in diagnostic assays.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This application relates to novel protein tyrosine kinases, thenucleic acid sequences encoding these proteins, the extracellulardomains of the proteins, ligands to the protein tyrosine kinases,antibodies specific for the encoded proteins and methods of usetherefor. In particular, this application relates to the novel receptorprotein tyrosine kinases designated Rse and HPTK6.

[0003] 2. Description of Related Art

[0004] Intracellular signals which control cell growth anddifferentiation are often mediated by tyrosine kinase proteins.

[0005] Tyrosine kinases catalyze protein phosphorylation using tyrosineas a substrate for phosphorylation. Members of the tyrosine kinasefamily can be recognized by the presence of several conserved amino acidregions in the tyrosine kinase catalytic domain (Hanks et al., Science:241: 42-52 [1988]). The tyrosine kinase domain is crucial for the signaltransduction pathways required for mitogenesis, transformation and celldifferentiation. Certain tyrosine kinases predominantly stimulate cellgrowth and differentiation, whereas other tyrosine kinases arrest growthand promote differentiation. Furthermore, depending on the cellularenvironment in which it is expressed, the same tyrosine kinase mayeither stimulate, or inhibit, cell proliferation (Schlessinger et al.,Neuron, 9: 383-391 [1992]).

[0006] Tyrosine kinase proteins can be classified as either receptortyrosine kinases or intracellular tyrosine kinases. Receptor tyrosinekinases (rPTKs) convey extracellular signals to intracellular signalingpathways thereby controlling cell proliferation and differentiation.These rPTKs share a similar architecture, with an intracellularcatalytic portion, a transmembrane domain and an extracellularligand-binding domain. (Schesslinger et al., supra). The extracellulardomains (ECDs), which are responsible for ligand binding andtransmission of biological signals, have been shown to be composed of anumber of distinct structural motifs. The intracellular domain comprisesa catalytic protein tyrosine kinase. The binding of ligand to theextracellular portion is believed to promote dimerization of the rPTKresulting in transphosphorylation and activation of the intracellulartyrosine kinase domain. In addition to their catalytic function, theintracellular domains (ICDs) of rPTKs may also serve as binding sitesfor other components of the signal transduction pathway. In particular,some proteins containing src-homology 2 (SH2) domains have been shown tointeract in a phosphorylation-dependent and sequence specific manner tospecific tyrosine residues within the ICD (Cantley et al., Cell, 64:281-302 [1991]).

[0007] A large number of protein tyrosine kinases have beencharacterized on the basis of their amino acid and nucleic acidsequences. For a review of these proteins see Hanks et al., supra.

[0008] Wo 93/15201 discloses isolation of several novel rPTK genes foundin human megakaryocytic and lymphocytic cells using degenerateoligonucleotide probes as primers in a polymerase chain reaction (PCR)to amplify tyrosine kinase DNA segments.

[0009] The recent publication by Johnson et al., Proc. Natl. Acad. Sci.,90: 5677-5681 (1993) discusses the characterization of a receptortyrosine kinase called discoidin domain receptor (i.e., DDR) which isabundantly expressed in breast carcinoma cell lines. DDR is consideredto have two features not found in other receptor tyrosine kinases.First, a region of the amino acid sequence near the N terminus of DDRcontains a “discoidin I-like domain”. This determination was based onthe sequence identity between this region and the protein, discoidin I(see FIG. 5 of Johnson et al.). Discoidin I-like domains are present astandem repeats at the C terminus of the light chains of factor V (Kane,W. H. & Davie, E. W., Proc. Natl. Acad. Sci., 83: 6800-6804 [1986]),factor VIII (Toole et al., Nature(London), 312: 342-347 [1984]) andVehar et al., Nature(London), 312: 337-342 [1984], and two milk fatglobule membrane proteins, MFG.E8 (see Stubbs et al., Proc. Natl. Acad.Sci., 87: 8417-8421 [1991]) and BA46 (see Larocca et al., Cancer Res.,51: 4994-4998 [1991]). Second, the DDR protein has an extensiveproline/glycine-rich region between the discoidin I-like domain and thetransmembrane domain and another such region between the transmembranedomain and the C-terminal tyrosine kinase domain. Theseproline/glycine-rich regions are not found in other receptor proteintyrosine kinases. The catalytic domain of DDR shares 45% sequenceidentity with the trk protein catalytic domain disclosed in Martin-Zancaet al., Mol. Cell. Biol., 9:24-33 (1989). Zerlin et al. discloseisolation of the murine equivalent of the DDR rPTK found by Johnson etal., which they call NEP (Oncogene, 8: 2731-2939 [1993]).

[0010] WO 92/14748 discloses a receptor, designated KDR, which isclassified as a type III receptor tyrosine kinase and binds to vascularendothelial cell growth factor. The type III group of rPTKs includes thec-kit proto-oncogene and the receptors for platelet derived growthfactor (PDGF) and colony stimulating factor-1 (CSF-1).

[0011] Matthews et al., Proc. Natl. Acad. Sci., 88: 9026-9030 (1991)refer to the isolation of rPTK clone from a population of hematopoieticmurine cells which, like KDR, exhibits a close sequence identity toc-kit. This receptor is called flk-1. The flk-1 receptor was isolatedusing an anti-sense oligonucleotide primer and anchored PCR. Chromosomalmapping indicated that the flk-1, kit and pdgfra genes are closelylinked. Matthews et al., Cell, 65: 1143-1152 (1991) discuss isolation ofa rPTK called flk-2, from stem cell-enriched murine hematopoietictissue. U.S. Pat. No. 5,185,438 also refers to the rPTKs, flk-1 andflk-2, which are said to be expressed in primitive hematopoietic cellsbut not in mature hematopoietic cells.

[0012] Lai et al., Neuron, 6: 691-704 (1991) used PCR to identifyseveral cDNAs encoding part of the tyrosine kinase domains of variousrat rPTKs. The newly isolated sequences were designated tyro-1 totyro-13. Because preferential expression of several of the sequences inthe developing vertebrate nervous system was evidenced, Lai et al.concluded that protein-tyrosine kinases appear to play a central role inneural development.

[0013] Holtrich et al., Proc. Natl. Acad. Sci., 88:10411-10415 (1991)studied the expression of protein-tyrosine-kinases in normal human lungand tumor cells by PCR followed by molecular cloning and sequenceanalysis. Six known PTKs (yes, fgr, lyn, hck, pdgfb-r and csfl-r) weredetected as well as two new PTKs. One of the proteins detected appearedto be cytosolic. The other PTK, designated TKF, was found to be relatedto fibroblast growth factor receptor and was only found expressed in thelung.

[0014] WO 93/14124 discloses the cloning, sequencing and expression of ahuman rPTK termed tie which is expressed in cultured endothelial cellsas well as tumor cell lines. The extracellular domain (ECD) of tie wasfound to contain stretches of amino acid sequence having features of theimmunoglobulin, epidermal growth factor and fibronectin type III repeatprotein families.

[0015] Partanen et al., Proc. Natl. Acad. Sci., 87: 8913-8917 (1990)analyzed PCR amplified cDNA clones which lead to the identification of14 different tyrosine kinase-related sequences, designated JTK1-14.Based on the pattern of expression of the clones, it was suggested thatthe tyrosine kinases encoded by the complete sequences most probablyplay a role in the differentiation of megakaryoblasts or in thephysiology of platelets.

[0016] While Partanen et al. discuss isolation of the partial JTK11 cDNAclone, the later publication by Janssen et al., Oncogene, 6: 2113-2120(1991), reports the cDNA cloning of the entire oncogene (designated UFO)encoding a 894 amino acid polypeptide. Janssen et al. identified the UFOtyrosine kinase receptor by DNA transfection analysis of bone marrowcells from a patient suffering from a chronic myeloproliferativedisorder. It is noted in this publication that several oncogene productsare rPTKs, e.g. colony-stimulating factor-1 and TRK. Around the sametime that Janssen et al. isolated the rPTK they call UFO, O'Bryan et al.isolated the same rPTK (which they designate Axl) from human myeloidleukemia cells (O'Bryan et al., Mol. Cell. Biol., 11: 5016-5031 [1991]).Axl is a transforming gene which encodes a rPTK having two fibronectintype III repeats and two immunoglobulin-like repeats in theextracellular domain thereof. These motifs are also found in theextracellular domain of the receptor-like protein tyrosine phosphatase,PTPμ (Brady-Kalnay et al., J. Cell Biol., 122: 961-972 [1993]). Theimmunoglobulin domain and four fibronectin type-III repeats of PTPμ aresimilar to the motifs found in cell-cell adhesion molecules.Brady-Kalnay et al. propose that the ligand for the PTPμ may be anotherPTPμ on an adjacent cell.

[0017] Faust et al., Oncogene, 7: 1287-1293 (1992) disclose cloning ofthe mouse homologue of the UFO oncogene identified in the publication byJanssen et al. This murine tyrosine kinase has an overall sequenceidentity of 87.6% with the human sequence. The extracellular domain ofthe UFO receptor is characterized by the existence of twoimmunoglobulin-like (IgL) and two fibronectin type III (FNIII) repeats.As discussed in Faust et al., a combination of IgL and FNIII domains arealso found in several neural cell adhesion molecules and receptortyrosine phosphatases suggesting that these structures are important forintercellular communication.

[0018] Wilks et al., Gene, 85: 67-74 (1989) used degenerateoligo-deoxyribonucleotide (oligo) primers derived from amino acidsequence motifs held in common between all members of the PTK family toprime the amplification of PTK sequences. It was found that the mosteffective type of primer for identification of PTK sequences is a short,moderately degenerate, oligo primer. Using the techniques disclosed,Wilks and his co-workers isolated a new mammalian PTK sequence as wellas other known PTK sequences.

[0019] Brauninger et al., Gene, 110(2): 205-211 (1992) discloseisolation of a human gene encoding an intracellular protein belonging toa new subclass of protein tyrosine kinases. The clone, designated csk,was found to be expressed in human lung and macrophages. The csk genewas distinguished from the src family of proto-oncogenes by the lack ofcertain tyrosine autophosphorylation sites in the amino acid sequenceand the lack of a N-terminal myristylation site.

[0020] It is evident that a number of rPTKs are involved in cell growthand differentiation, many of which have been characterized to date.

[0021] Additional rPTKs are needed in order to further study growth anddifferentiation of cells, for use as therapeutic agents and fordiagnostic use.

[0022] Accordingly, it is an object of this invention to identify andpurify one or more novel protein tyrosine kinase receptors. It is yetanother object to provide derivatives and modified forms of such newpolypeptides, including amino acid sequence variants and covalentderivatives thereof.

[0023] It is another object to provide nucleic acid encoding such novelrPTKs and to use this nucleic acid to produce rPTKs in recombinant cellculture. The rPTK protein thus produced can be used for investigational,therapeutic or diagnostic use. Nucleic acid sequences which hybridizewith the DNA or RNA encoding the proteins described herein can also beused as anti-sense oligonucleotides to inhibit protein tyrosine kinaseactivity either in vivo or in vitro.

[0024] It is a further object to provide amino acid sequences encodingthe ECDs of the novel rPTKs, which sequences are useful for in vitroassays or for use as therapeutic agents. The ECDs, or variants thereof,can also be used as immunogens for raising antibodies, including agonistantibodies to the rPTKs. Nucleic acid sequences encoding the novel rPTKECDs are needed in order to make these polypeptides recombinantly.

[0025] Ligands to the novel rPTKs are also desirable for use astherapeutic agents to stimulate the receptor and thereby stimulate cellgrowth and/or differentiation. Such ligands are useful for determiningthe function and biological activity of the receptors.

[0026] These and other objects will be apparent to the ordinary artisanupon consideration of the specification as a whole.

SUMMARY OF THE INVENTION

[0027] These objects are accomplished, in one aspect, by providingisolated Rse or HPTK6 rPTKs that may be antigenically or biologicallyactive.

[0028] In another aspect, the invention provides a compositioncomprising biologically active Rse or HPTK6 and a pharmaceuticallyacceptable carrier.

[0029] According to another object of the invention, the isolatedextracellular domains of each of the novel rPTKs are provided which canbe used to raise antibodies against each of the novel rPTKs.

[0030] In another aspect, the invention provides isolated ligands whichbind to the extracellular domain of the rPTKs. Such ligands can act asantagonists or agonists and thereby either stimulate, or inhibit,tyrosine kinase activity of the rPTKs.

[0031] The invention also provides isolated nucleic acid sequencesencoding the entire rPTK amino acid sequence or the extracellular domainthereof, as well as nucleic acid sequences encoding protein ligands tothe novel rPTK proteins.

[0032] In still further aspects, the nucleic acid is provided in areplicable vector comprising the nucleic acid encoding the proteinsdisclosed. The invention also provides host cells transformed with thevector. A method of using the nucleic acid encoding the proteins toeffect the production of the novel proteins is also provided whichcomprises expressing the nucleic acid in a culture of the transformedhost cells and recovering the protein from the host cell culture.

[0033] In further embodiments, the invention provides a method ofenhancing cell growth or differentiation comprising administering to amammalian patient in need of such treatment an exogenous compoundselected from the group consisting of: Rse rPTK; HPTK6 rPTK; agonistligand to Rse rPTK; and agonist ligand to HPTK6 rPTK, in an amounteffective in inducing cell growth or differentiation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1A depicts the nucleic acid sequence for human Rse (SEQ IDNO: 1) and the deduced amino acid sequence thereof (SEQ ID NO: 2). Thenucleic acid sequence of the extracellular domain of Rse (SEQ ID NO: 5)and the amino acid sequence of the extracellular domain of Rse (SEQ IDNO: 6) are indicated by dots. FIG. 1B depicts the nucleic acid sequencefor murine Rse (SEQ ID NO: 9) and the deduced amino acid sequencethereof (SEQ ID NO: 10). The nucleic acid sequence of the extracellulardomain (SEQ ID NO: 11) and the amino acid sequence of the extracellulardomain thereof (SEQ ID NO: 12) are indicated by dots.

[0035] In FIGS. 1A and 1B, the composite nucleic acid sequencedetermined from overlapping cDNA clones is shown on the bottom line. Thetranslated sequence, in single-letter amino acid code, is shown on thetop line. The predicted signal sequences are printed in bold-type, andthe potential sites for N-linked glycosylation are indicated with an(*). The putative transmembrane domains are boxed. The arrows delineatethe start and end of the putative tyrosine kinase domain, and withinthat domain, the consensus sites for Mg²⁺-ATP binding (beginning atamino acids 525 and 515 of the human and murine Rse proteins,respectively) and the region often used to predict substrate specificity(beginning at amino acids 652 and 642 of the human and murine Rseproteins, respectively) are underlined. Human and murine Rse sequencesrepresent a total of 3,611 and 3,785 nucleotides, respectively,determined from overlapping clones sequenced in both directions. Thehuman Rse cDNA S sequence ends at an internal EcoRI site in the 3′untranslated region; the murine Rse cDNA includes the polyadenylationsequence.

[0036]FIG. 2 depicts the nucleic acid sequence for human HPTK6 (SEQ IDNO: 3) and the deduced amino acid sequence thereof (SEQ ID NO: 4). Thenucleic acid sequence of the extracellular domain of HPTK6 (SEQ ID NO:7) and the amino acid sequence of the extracellular domain of HPTK6 (SEQID NO: 8) are in bold, the putative transmembrane domain is boxed, theamino acid residues forming the signal sequence are indicated with an(*) and the putative ATP binding site in the kinase domain is indicatedby dots. The arrows delineate the start and end of the putative tyrosinekinase domain.

[0037]FIG. 3 is a diagrammatic representation of the structural domainsand hydrophobicity plot of human and murine Rse. A schematicrepresentation of the immunoglobulin-like (IgL) domains, fibronectintype III domains (FNIII), transmembrane domain (TM) and tyrosine kinase(Kinase) domains of Rse is shown on the top line. Below, thehydrophobicity profile of human Rse and murine Rse is shown. The HYDROprogram (Genentech, Inc.) was used to obtain the hydrophobicity plots.

[0038]FIG. 4 depicts a comparison of the amino acid sequences of humanand murine Rse (i.e., hRSE and mRSE, SEQ ID NOS: 2 and 10,respectively), and Axl (i.e., hAXL and mAXL, SEQ ID NOS: 34 and 35,respectively). Sequences were aligned using the ALIGN program. Gapsintroduced for optimal alignment are indicated by dots. The amino acidpositions are numbered from the initiation methionine. Conservedresidues are boxed. Immunoglobulin-like domains 1 and 2 (IgL-1 andIgL-2), fibronectin type III-like domains 1 and 2 (FN-1 and FN-2), andthe tyrosine kinase homology region are indicated. The highly conservedamino acids in the IgL domains are indicated by (*), and the elevenhighly conserved domains (Hanks et al., supra) within the tyrosinekinase region are marked.

[0039]FIG. 5 illustrates expression and activation of gD-Rse. Totallysates from NIH3T3 cells (lanes 1, 3, 5, 7, 9, and 11) or 3T3.gD.R11cells (lanes 2, 4, 6, 8, 10, and 12) were immunoprecipitated with theantibody 5B6 which detects the gD portion of the fusion protein, and theimmunoprecipitates were resolved by SDS-PAGE and immunoblotted withanti-phosphotyrosine antibodies (lanes 7-12). After the blots weredeveloped, they were stripped and re-probed with antibody 5B6 (lanes1-6). Lysates were prepared from cells grown in the absence of addedantibody (lanes 1, 2, 7, and 8) or incubated with antibody 5B6 (lanes 3,4, 9, and 10) or an isotype-matched control antibody A3.1.2 (lanes 5, 6,11, and 12). Molecular masses (kDa) are indicated on the right.

[0040]FIG. 6 depicts a time course of antibody induced stimulation ofgD-Rse tyrosine kinase activity. 3T3.gD.R11 cells were incubated without(−) or with antibody 5B6 for 10, 30, 60, or 120 minutes (Min.) asindicated. Western blots were prepared as described for FIG. 5. The blotwas reacted first with the anti-phosphotyrosine antibody 5E2 (α-pTyr)then stripped and reacted with antibody 5B6 (∝-gD) to control for theamount of gD-Rse on the blots.

[0041]FIGS. 7A and 7B show a Northern blot analysis of Rse mRNAexpression in adult human tissues. In FIG. 7A, a Northern blotcontaining 2 μg of poly(A) RNA isolated from human tissues washybridized to a ³²P-labeled probe corresponding to human Rse nucleotides195-680 (FIG. 1A). Positions of markers are indicated on the right inKb. Lane 1: heart, lane 2: brain, lane 3: placenta, lane 4: lung, lane5: liver, lane 6: skeletal muscle, lane 7: kidney, lane 8: pancreas. InFIG. 7B, the blot shown in FIG. 7A was washed and then hybridized with a³²P-labeled beta-actin probe to confirm the integrity of the RNAsamples.

[0042]FIGS. 8A and 8B depict the chromosomal localization of the humanRse gene. FIG. 8A depicts the ethidium-stained PCR product of one of twoamplifications using independent primer sets (Btk 3-1²⁷²⁴, Btk 3-4),corresponding to unique 3′-untranslated sequences in Rse amplifyinggenomic DNA derived from a panel of human-CHO hybrid cell lines (lanes1-25), human control (lane 26) or hamster control (lane 27). In FIG. 8B,the matrix of hybrid cell line number and corresponding human chromosomeis highlighted to indicate the match of the PCR product with humanchromosome 15.

[0043]FIG. 9 depicts stimulation of gD-Rse by polyclonal antibodies.Immunoprecipitates from control NIH3T3 cells (lanes 1 and 3) or3T3.gD.R11 cells (2 and 4) were prepared using the anti-gD antibody 5B6,resolved by SDS-PAGE and immunoblotted with antiphosphotyrosineantibodies. Cells were either untreated (lanes 1 and 2) or treated(lanes 3 and 4) for 10 minutes with rabbit polyclonal antiserum preparedagainst a fusion protein containing the extracellular domain of Rse.

[0044]FIGS. 10A and 10B show a Northern blot analysis of HPTK6 mRNAexpression in adult (FIG. 10A) and fetal (FIG. 10B) human tissues. ANorthern blot containing 2 μg of poly(A) RNA isolated from human tissueswas hybridized to a ³²P-labeled probe corresponding to human HPTK6nucleotides 11-622 (FIG. 2). Positions of markers are indicated on theleft in Kb.

[0045]FIGS. 11A and 11B show a Northern blot analysis of HPTK6 mRNAexpression in adult mouse tissue. In FIG. 11A, a Northern blotcontaining 2 μg of poly(A) RNA isolated from human tissues washybridized to a ³²P-labeled probe corresponding to human HPTK6nucleotides 11-622 (FIG. 2). Positions of markers are indicated on theleft in Kb. For FIG. 11B, the blot shown in FIG. 11A was washed and thenhybridized with a ³²P-labeled beta-actin probe to confirm the integrityof the RNA samples.

[0046]FIGS. 12A, 12B, and 12C depict in situ hybridization of HPTK6 inhuman (FIG. 12A) and mouse (FIGS. 12B and 12C) fetal tissue. Transversesection through human or mouse embryos were hybridized with ³²P-labeledantisense (−ve) and sense (tve) strands.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

[0047] In general, the following words or phrases have the indicateddefinition when used in the description, examples, and claims:

[0048] “Receptor Protein Tyrosine Kinases” (rPTKs), when used throughoutthe detailed description of the invention, refers to Rse and HPTK6proteins. It also refers to both full-sequence and ECD unlessspecifically stated otherwise.

[0049] “Rse” is defined herein to be any polypeptide sequence thatpossesses a biological property of a naturally occurring polypeptidecomprising the polypeptide sequence of FIG. 1A or 1B.

[0050] “HPTK6” is defined herein to be any polypeptide sequence thatpossesses a biological property of a naturally occurring polypeptidecomprising the polypeptide sequence of FIG. 2.

[0051] “Biological property” for the purposes herein means an in vivoeffector or antigenic function or activity that is directly orindirectly performed by Rse or HPTK6 (whether in its native or denaturedconformation). Effector functions include receptor function, ligandbinding, signal transduction, phosphorylation using tyrosine as asubstrate for phosphorylation, dimerization of the rPTK resulting intransphosphorylation and activation of the catalytic kinase domain, anyenzyme activity or enzyme modulatory activity (e.g., tyrosine kinaseactivity), stimulation of cell growth and/or differentiation, inhibitionof cell growth or proliferation, or any structural role. However,effector functions do not include possession of an epitope or antigenicsite that is capable of cross-reacting with antibodies raised againstRse or HPTK6. An antigenic function means possession of an epitope orantigenic site that is capable of cross-reacting with antibodies raisedagainst the polypeptide sequence of a naturally occurring polypeptidecomprising the polypeptide sequence of FIG. 1A, 1B or FIG. 2.

[0052] “Biologically active” rPTK is defined herein as a polypeptidethat shares an effector function of rPTK and that may (but need not) inaddition possess an antigenic function. A principal known effectorfunction of rPTK is its ability to catalyze protein phosphorylationusing tyrosine as a substrate for phosphorylation. The biologicalactivity of rPTK may be further characterized by its ability tostimulate cell growth or differentiation in vivo or in vitro.

[0053] “Antigenically active” rPTK is defined as a polypeptide thatpossesses an antigenic function of rPTK and that may (but need not) inaddition possess an effector function.

[0054] In preferred embodiments, antigenically active rPTK is apolypeptide that binds with an affinity of at least about 10⁶ l/mole toan antibody capable of binding rPTK. Ordinarily, the polypeptide bindswith an affinity of at least about 10⁷ l/mole. Isolated antibody capableof binding rPTK is an antibody that is identified and separated from acomponent of the natural environment in which it may be present. Mostpreferably, the antigenically active rPTK is a polypeptide that binds toan antibody capable of binding rPTK in its native conformation. rPTK inits native conformation is rPTK as found in nature that has not beendenatured by chaotropic agents, heat, or other treatment thatsubstantially modifies the three-dimensional structure of rPTK asdetermined, for example, by migration on non-reducing, non-denaturingsizing gels. Antibody used in this determination is rabbit polyclonalantibody raised by formulating native rPTK from a non-rabbit species inFreund's complete adjuvant, subcutaneously injecting the formulation,and boosting the immune response by intraperitoneal injection of theformulation until the titer of anti-rPTK antibody plateaus.

[0055] Ordinarily, biologically or antigenically active rPTK will havean amino acid sequence having at least 75% amino acid sequence identitywith the mature rPTK amino acid sequence shown in either FIG. 1A, 1B orFIG. 2, more preferably at least 80%, more is preferably at least 85%,more preferably at least 90%, and most preferably at least 95%. Identityor homology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the rPTK residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the rPTK sequence shall beconstrued as affecting sequence identity or homology.

[0056] Thus, the biologically active and antigenically active rPTKpolypeptides that are the subject of this invention include thepolypeptide represented by the entire translated nucleotide sequence ofrPTK; mature rPTK; fragments thereof having a consecutive sequence of atleast 5, 10, 15, 20, 25, 30, or 40 amino acid residues from rPTK; aminoacid sequence variants of rPTK wherein an amino acid residue has beeninserted N- or C-terminal to, or within, rPTK or its fragment as definedabove; amino acid sequence variants of rPTK or its fragment as definedabove wherein an amino acid residue of rPTK or its fragment as definedabove has been substituted by another residue, including predeterminedmutations by, e.g., site-directed or PCR mutagenesis, rPTK of variousanimal species-such as rabbit, rat, porcine, non-human primate, equine,murine, and ovine rPTK and alleles or other naturally occurring variantsof the foregoing and human rPTK; derivatives of rPTK or its fragments asdefined above wherein rPTK or its fragments have been covalent modified,by substitution, chemical, enzymatic, or other appropriate means, with amoiety other than a naturally occurring amino acid; and glycosylationvariants of rPTK (insertion of a glycosylation site or alteration of anyglycosylation site by deletion, insertion, or substitution of suitableresidues). Such fragments and variants exclude any polypeptideheretofore identified, including any known rPTK of any animal species orany known polypeptide fragment, which is anticipatory under 35 USC §102as well as polypeptides obvious thereover under 35 USC §103. Thepreferred rPTK is human mature rPTK.

[0057] An “exogenous” therapeutic compound is defined herein to mean atherapeutic compound that is foreign to the mammalian patient, orhomologous to a compound found in the mammalian patient but producedoutside the mammalian patient.

[0058] “Extracellular domain” (ECD) of rPTK is defined herein to be anypolypeptide sequence that shares a ligand binding function of the ECD ofthe naturally occurring Rse polypeptide shown in FIG. 1A or 1B; or theECD of the naturally occurring HPTK6 polypeptide shown in FIG. 2 andthat may (but need not) in addition possess an antigenic function of thenative extracellular domain of Rse or HPTK6. Ligand binding function ofthe ECD refers to the ability of the polypeptide to bind at least oneRse ligand or at least one HPTK6 ligand. An antigenic function of theECD means possession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against the polypeptide sequenceof a naturally occurring polypeptide comprising the polypeptide sequenceof the ECD of Rse or HPTK6 shown in FIG. 1A, 1B or FIG. 2. The ECD isessentially free of the transmembrane and intracellular domains ofnative Rse or HPTK6, i.e., has less than 1% of such domains, preferably0.5 to 0 of such domains, and more preferably 0.1 to 0% of such domains.

[0059] Ordinarily, the rPTK ECD will have an amino acid sequence havingat least 75% amino acid sequence identity with the amino acid sequenceof the ECD of Rse indicated in FIG. 1A or 1B, or the ECD of HPTK6indicated in FIG. 2, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, and most preferably at least95%.

[0060] Thus, the ECDs of Rse or HPTK6 that are the subject of thisinvention include the polypeptide represented by the entire LStranslated nucleotide sequence of the ECD of Rse or HPTK6; amino acidsequence variants of the ECD of Rse or HPTK6 wherein an amino acidresidue has been inserted N- or C-terminal to, or within the ECD; aminoacid sequence variants of the ECD wherein an amino acid residue of thenative ECD of Rse or HPTK6 has been substituted by another residue,including predetermined mutations by, e.g., site-directed or PCRmutagenesis, the ECD of Rse or HPTK6 of various animal species such asrabbit, rat, porcine, non-human primate, equine, murine, and ovine rPTKECD and alleles or other naturally occurring variants of the foregoingand human ECDS; derivatives of the ECD wherein the ECD has beencovalently modified, by substitution, chemical, enzymatic, or otherappropriate means, with a moiety other than a naturally occurring aminoacid; any glycosylation variants of the ECD. Such variants exclude anypolypeptide heretofore identified, which is anticipatory under 35 USC§102 as well as polypeptides obvious thereover under 35 USC §103. Thepreferred rPTK ECD is the ECD of human Rse or HPTK6.

[0061] “Ligand”, when used herein, is defined to encompass any molecule,protein or non-protein, which is able to bind to the ECD of Rse orHPTK6. The ligand may be an agonist or an antagonist to Rse or HPTK6.Generally, the ligand will activate one of the effector functions of therPTK. For example, upon binding the ECD of the rPTK, the ligand maystimulate tyrosine kinase activity. Stimulation of tyrosine kinaseactivity may, for example, be caused by dimerization of the rPTK whichresults in transphosphorylation of the kinase domain. Consequently,binding of the ligand to the receptor may result in an enhancement ofcell growth and/or differentiation in vivo or in vitro or, conversely,cell growth may be arrested and cell differentiation may be stimulatedupon binding of the ligand to the receptor. The ligand may be theendogenous ligand for the receptor and will generally be a polypeptide.In one embodiment disclosed herein, the ligand is an antibody againstthe ECD of the rPTK. The preferred antibody is a humanized monoclonalantibody against the ECD of rPTK. A “humanized” antibody is a chimericantibody wherein substantially less than an intact human variable domainhas been substituted by the corresponding sequence from a non-humanspecies. Such ligands exclude any molecule heretofore identified, whichis anticipatory under 35 USC §102 as well as any molecule obviousthereover under 35 USC §103. The preferred ligand is the endogenousligand to the ECD of Rse or HPTK6.

[0062] “Isolated”, when used to describe the various proteins disclosedherein, means protein that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the protein, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the protein will be purified (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (2) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated protein includesprotein in situ within recombinant cells, since at least one componentof the rPTK natural environment will not be present. Ordinarily,however, isolated protein will be prepared by at least one purificationstep.

[0063] “Essentially pure” protein means a composition comprising atleast about 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight. “Essentiallyhomogeneous” protein means a composition comprising at least about 99%by weight of protein, based on total weight of the composition.

[0064] In accordance with this invention, rPTK nucleic acid or a rPTKnucleic acid molecule is RNA or DNA containing greater than ten basesthat encodes a biologically active or antigenically active rPTK, iscomplementary to nucleic acid sequence encoding such rPTK, or hybridizesto nucleic acid sequence encoding such rPTK and remains stably bound toit under stringent conditions. The nucleic acid encoding the rPTKs,comprises nucleic acid residue nos 7-2676 of FIG. 1A (i.e., hRse nucleicacid); nucleic acid residue nos 62-2701 of FIG. 1B (i.e., mRse nucleicacid); or nucleic acid residue nos 82-2820 of FIG. 2 (i.e., HPTK6nucleic acid). In one embodiment, the nucleic acid sequence is selectedfrom (a) the nucleic acid sequences of FIG. 1A, 1B or FIG. 2, (b) asequence corresponding to the sequences of (a) within the scope ofdegeneracy of the genetic code or (c) a sequence which hybridizes with asequence defined in (a) or (b) above under stringent conditions.

[0065] Preferably, the rPTK nucleic acid molecule encodes a polypeptidesharing at least 75% sequence identity, more preferably at least 80%,still more preferably at least 85%, even more preferably at least 90%,and most preferably 95%, with the rPTK amino acid sequence shown in FIG.1A, 1B or FIG. 2.

[0066] Preferably, the rPTK nucleic acid molecule that hybridizes tonucleic acid sequence encoding rPTK contains at least 20, morepreferably 40, and most preferably 90 bases. Such hybridizing orcomplementary nucleic acid molecule, however, is further defined asbeing novel under 35 USC §102 and unobvious under 35 USC §103 over anyprior art nucleic acid molecules.

[0067] Stringent conditions are those that (1) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1 NaDodSO₄ at 50° C.; (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

[0068] An isolated rPTK nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the rPTK nucleic acid. An isolated rPTK nucleic acid moleculeis other than in the form or setting in which it is found in nature.Isolated rPTK nucleic acid molecules therefore are distinguished fromthe rPTK nucleic acid molecule as it exists in natural cells. However,an isolated rPTK nucleic acid molecule includes rPTK nucleic acidmolecules contained in cells that ordinarily express rPTK where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

[0069] In accordance with this invention, rPTK ECD nucleic acid or arPTK nucleic acid molecule encoding the ECD of rPTK is RNA or DNAcontaining greater than ten bases that encodes a polypeptide that sharesa ligand binding function of Rse ECD or HPTK6 ECD and that may (but neednot) in addition possess an antigenic function, is complementary tonucleic acid sequence encoding such ECD, or hybridizes to nucleic acidsequence encoding such ECD and remains stably bound to it understringent conditions. In one embodiment, the nucleic acid sequence isselected from (a) the nucleic acid sequences of SEQ ID NO; 5, SEQ ID NO:7 or SEQ ID NO: 11, (b) a sequence corresponding to the sequencesdefined in (a) within the scope of degeneracy of the genetic code or (c)a sequence which hybridizes with a sequence defined in (a) or (b) aboveunder stringent conditions.

[0070] Preferably, the rPTK ECD nucleic acid molecule encodes apolypeptide sharing at least 75% sequence identity, more preferably atleast 80%, still more preferably at least 85%, even more preferably atleast 90%, and most preferably 95%, with the amino 0.5 acid sequences ofSEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 11.

[0071] Such hybridizing or complementary nucleic acid molecule, however,is further defined as being novel under 35 USC §102 and unobvious under35 USC §103 over any prior art nucleic acid molecules.

[0072] The isolated rPTK polypeptide or rPTK nucleic acid may be labeledfor diagnostic and probe purposes, using a label as described anddefined further below in the discussion of diagnostic assays.

[0073] The expression “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and possibly, other as yet poorlyunderstood sequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0074] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

[0075] As used herein, the expressions “cell,” “cell line,” and “cellculture” are used interchangeably and all such designations includeprogeny. Thus, the words “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

[0076] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare commercially available, are publicly available on an unrestrictedbasis, or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

[0077] “Restriction enzyme digestion” of DNA refers to catalyticcleavage of the DNA with an enzyme that acts only at certain locationsin the DNA. Such enzymes are called restriction endonucleases, and thesite for which each is specific is called a restriction site. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors, and other requirements asestablished by the enzyme suppliers are used. Restriction enzymescommonly are designated by abbreviations composed of a capital letterfollowed by other letters representing the microorganism from which eachrestriction enzyme originally was obtained and then a number designatingthe particular enzyme. In general, about 1 μg of plasmid or DNA fragmentis used with about 1-2 units of enzyme in about 20 μl of buffersolution. Appropriate buffers and substrate amounts for particularrestriction enzymes are specified by the manufacturer. Incubation ofabout 1 hour at 37° C. is ordinarily used, but may vary in accordancewith the supplier's instructions. After incubation, protein orpolypeptide is removed by extraction with phenol and chloroform, and thedigested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzyme may befollowed with bacterial alkaline phosphatase hydrolysis of the terminal5′ phosphates to prevent the two restriction-cleaved ends of a DNAfragment from “circularizing” or forming a closed loop that would impedeinsertion of another DNA fragment at the restriction site. Unlessotherwise stated, digestion of plasmids is not followed by 5′ terminaldephosphorylation. Procedures and reagents for dephosphorylation areconventional as described in sections 1.56-1.61 of Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989).

[0078] “Recovery” or “isolation” of a given fragment of DNA from arestriction digest means separation of the digest on polyacrylamide oragarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA.This procedure is known generally. For example, see Lawn et al., NucleicAcids Res., 9: 6103-6114 (1981), and Goeddel et al., Nucleic AcidsRes.8: 4057 (1980).

[0079] “Southern analysis” is a method by which the presence of DNAsequences in a restriction endonuclease digest of DNA or DNA-containingcomposition is confirmed by hybridization to a known, labeledoligonucleotide or DNA fragment. Southern analysis typically involveselectrophoretic separation of DNA digests on agarose gels, denaturationof the DNA after electrophoretic separation, and transfer of the DNA tonitrocellulose, nylon, or another suitable membrane support for analysiswith a radiolabeled, biotinylated, or enzyme-labeled probe as describedin sections 9.37-9.52 of Sambrook et al., supra.

[0080] “Northern analysis” is a method used to identify RNA sequencesthat hybridize to a known probe such as an oligonucleotide, DNAfragment, cDNA or fragment thereof, or RNA fragment. The probe islabeled with a radioisotope such as ³²P, or by biotinylation, or with anenzyme. The RNA to be analyzed is usually electrophoretically separatedon an agarose or polyacrylamide gel, transferred to nitrocellulose,nylon, or other suitable membrane, and hybridized with the probe, usingstandard techniques well known in the art such as those described insections 7.39-7.52 of Sambrook et al., supra.

[0081] “Ligation” refers to the process of forming phosphodiester bondsbetween two nucleic acid fragments. For ligation of the two fragments,the ends of the fragments must be compatible with each other. In somecases, the ends will be directly compatible after endonucleasedigestion. However, it may be necessary first to convert the staggeredends commonly produced after endonuclease digestion to blunt ends tomake them compatible for ligation. For blunting the ends, the DNA istreated in a suitable buffer for at least 15 minutes at 15° C. withabout 10 units of the Klenow fragment of DNA polymerase I or T4 DNApolymerase in the presence of the four deoxyribonucleotidetriphosphates. The DNA is then purified by phenol-chloroform extractionand ethanol precipitation. The DNA fragments that are to be ligatedtogether are put in solution in about equimolar amounts. The solutionwill also contain ATP, ligase buffer, and a ligase such as T4 DNA ligaseat about 10 units per 0.5 μg of DNA. If the DNA is to be ligated into avector, the vector is first linearized by digestion with the appropriaterestriction endonuclease(s). The linearized fragment is then treatedwith bacterial alkaline phosphatase or calf intestinal phosphatase toprevent self-ligation during the ligation step.

[0082] “Preparation” of DNA from cells means isolating the plasmid DNAfrom a culture of the host cells. Commonly used methods for DNApreparation are the large- and small-scale plasmid preparationsdescribed in sections 1.25-1.33 of Sambrook et al., supra. Afterpreparation of the DNA, it can be purified by methods well known in theart such as that described in section 1.40 of Sambrook et al., supra.

[0083] “Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid-phase techniques such as described in EP 266,032 published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14: 5399-5407 (1986). Further methodsinclude the polymerase chain reaction defined below and other autoprimermethods and oligonucleotide syntheses on solid supports. All of thesemethods are described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989). These methods may be used if the entire nucleic acidsequence of the gene is known, or if the sequence of the nucleic acidcomplementary to the coding strand is available. Alternatively, if thetarget amino acid sequence is known, one may infer potential nucleicacid sequences using known and preferred coding residues for each aminoacid residue. The oligonucleotides are then purified on polyacrylamidegels.

[0084] The technique of “polymerase chain reaction,” or “PCR,” as usedherein generally refers to a procedure wherein minute amounts of aspecific piece of nucleic acid, RNA and/or DNA, are amplified asdescribed in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987. Generally,sequence information from the ends of the region of interest or beyondneeds to be available, such that oligonucleotide primers can bedesigned; these primers will be identical or similar in sequence toopposite strands of the template to be amplified. The 5′ terminalnucleotides of the two primers may coincide with the ends of theamplified material. PCR can be used to amplify specific RNA sequences,specific DNA sequences from total genomic DNA, and cDNA transcribed fromtotal cellular RNA, bacteriophage or plasmid sequences, etc. Seegenerally Mullis et al., Cold Spring is Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Fora recent review on PCR advances, see Erlich et al., Science, 252:1643-1650 (1991).

[0085] As used herein, PCR is considered to be one, but not the only,example of a nucleic acid polymerase reaction method for amplifying anucleic acid test sample comprising the use of a known nucleic acid as aprimer and a nucleic acid polymerase to amplify or generate a specificpiece of nucleic acid.

II. Modes for Practicing the Invention

[0086] Section 1 which follows, provides methodologies for preparingfull sequence rPTK, rPTK ECD, polypeptide ligands and variants thereof.The techniques disclosed in this section can be utilized for themanufacture of polypeptide ligands to the Rse and HPTK6 receptors.

[0087] 1. Preparation of Natural Sequence rPTK and Variants Thereof

[0088] Most of the discussion below pertains to production of rPTK byculturing cells transformed with a vector containing rPTK nucleic acidand recovering the polypeptide from the cell culture. It is furtherenvisioned that the rPTK of this invention may be produced by homologousrecombination, as provided for in WO 91/06667 published 16 May 1991.Briefly, this method involves transforming primary mammalian cellscontaining endogenous rPTK gene (e.g., human cells if the desired rPTKis human) with a construct (i.e., vector) comprising an amplifiable gene[such as dihydrofolate reductase, (DHFR) or others discussed below] andat least one flanking region of a length of at least about 150 bp thatis homologous with a DNA sequence at the locus of the coding region ofthe rPTK gene to provide amplification of the rPTK gene. The amplifiablegene must be at a site that does not interfere with expression of therPTK gene. The transformation is conducted such that the constructbecomes homologously integrated into the genome of the primary cells todefine an amplifiable region.

[0089] Primary cells comprising the construct are then selected for isby means of the amplifiable gene or other marker present in theconstruct. The presence of the marker gene establishes the presence andintegration of the construct into the host genome. No further selectionof the primary cells need be made, since selection will be made in thesecond host. If desired, the occurrence of the homologous recombinationevent can be determined by employing PCR and either sequencing theresulting amplified DNA sequences or determining the appropriate lengthof the PCR fragment when DNA from correct homologous integrants ispresent and expanding only those cells containing such fragments. Alsoif desired, the selected cells may be amplified at this point bystressing the cells with the appropriate amplifying agent (such asmethotrexate if the amplifiable gene is DHFR), so that multiple copiesof the target gene are obtained. Preferably, however, the amplificationstep is not conducted until after the second transformation describedbelow.

[0090] After the selection step, DNA portions of the genome,sufficiently large to include the entire amplifiable region, areisolated from the selected primary cells. Secondary mammalian expressionhost cells are then transformed with these genomic DNA portions andcloned, and clones are selected that contain the amplifiable region. Theamplifiable region is then amplified by means of an amplifying agent ifnot already amplified in the primary cells. Finally, the secondaryexpression host cells now comprising multiple copies of the amplifiableregion containing rPTK are grown so as to express the gene and producethe protein.

[0091] A. Isolation of DNA Encoding rPTK

[0092] The DNA encoding rPTK may be obtained from any cDNA libraryprepared from tissue believed to possess the rPTK mRNA and to express itat a detectable level. Accordingly, Rse can be conveniently obtainedfrom a cDNA library prepared from human brain or kidney tissue and HPTK6can be obtained from a cDNA library prepared from human adult kidneytissue. The rPTK gene may also be obtained from a genomic library or byoligonucleotide synthesis as defined above assuming the completenucleotide or amino acid sequence is known.

[0093] Libraries are screened with probes designed to identify the geneof interest or the protein encoded by it. For cDNA expression libraries,suitable probes include monoclonal or polyclonal antibodies thatrecognize and specifically bind to the rPTK; oligonucleotides of about20-80 bases in length that encode known or suspected portions of therPTK cDNA from the same or different species; and/or complementary orhomologous cDNAs or fragments thereof that encode the same or a similargene. Appropriate probes for screening genomic DNA libraries include,but are not limited to, oligonucleotides, cDNAs, or fragments thereofthat encode the same or a similar gene, and/or homologous genomic DNAsor fragments thereof. Screening the cDNA or genomic library with theselected probe may be conducted using standard procedures as describedin chapters 10-12 of Sambrook et al., supra.

[0094] An alternative means to isolate the gene encoding rPTK is to usePCR methodology as described in section 14 of Sambrook et al., supra.This method requires the use of oligonucleotide probes that willhybridize to the rPTK. Strategies for selection of oligonucleotides aredescribed below.

[0095] A preferred method of practicing this invention is to usecarefully selected oligonucleotide sequences to screen cDNA librariesfrom various tissues, preferably mammalian brain and kidney cell lines,more preferably, human brain and human kidney cell lines.

[0096] The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The actual nucleotide sequence(s) is usually based onconserved or highly homologous nucleotide sequences or regions of otherprotein tyrosine kinase molecules. The oligonucleotides may bedegenerate at one or more positions. The use of degenerateoligonucleotides may be of particular importance where a library isscreened from a species in which preferential codon usage is not known.

[0097] The oligonucleotide must be labeled such that it can be detectedupon hybridization to DNA in the library being screened. The preferredmethod of labeling is to use ³²P labeled ATP with polynucleotide kinase,as is well known in the art, to radiolabel the oligonucleotide. However,other methods may be used to label the oligonucleotide, including, butnot limited to, biotinylation or enzyme labeling.

[0098] Of particular interest is the rPTK nucleic acid that encodes afull-length polypeptide. In some preferred embodiments, the nucleic acidsequence includes the native rPTK signal sequence. Nucleic acid havingall the protein coding sequence is obtained by screening selected cDNAor genomic libraries using the deduced amino acid sequence disclosedherein for the first time, and, if necessary, using conventional primerextension procedures as described in section 7.79 of Sambrook et al.,supra, to detect precursors and processing intermediates of mRNA thatmay not have been reverse-transcribed into cDNA.

[0099] B. Amino Acid Sequence Variants of Native rPTK

[0100] Amino acid sequence variants of rPTK are prepared by introducingappropriate nucleotide changes into the rPTK DNA, or by synthesis of thedesired rPTK polypeptide. Such variants include, for example, deletionsfrom, or insertions or substitutions of, residues within the amino acidsequences shown for the rPTKs in FIGS. 1A, 1B & 2. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. Excluded from the scope of this invention are rPTKvariants or polypeptide sequences that are not novel and unobvious overthe prior art. The amino acid changes also may alter post-translationalprocesses of the rPTK, such as changing the number or position ofglycosylation sites, altering the membrane anchoring characteristics,and/or altering the intracellular location of the rPTK by inserting,deleting, or otherwise affecting the leader sequence of the rPTK.

[0101] For the design of amino acid sequence variants of rPTK, thelocation of the mutation site and the nature of the mutation will dependon the rPTK characteristic(s) to be modified. The sites for mutation canbe modified individually or in series, e.g., by (1) substituting firstwith conservative amino acid choices and then with more radicalselections depending upon the results achieved, (2) deleting the targetresidue, or (3) inserting residues of the same or a different classadjacent to the located site, or combinations of options 1-3.

[0102] A useful method for identification of certain residues or regionsof the rPTK polypeptide that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells, Science, 244: 1081-1085 (1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed rPTK variants are screened for the optimal combination ofdesired activity.

[0103] There are two principal variables in the construction of aminoacid sequence variants: the location of the mutation site and the natureof the mutation. These are variants of the sequences of FIGS. 1A, 1B &2, and may represent naturally occurring alleles (which will not requiremanipulation of the rPTK DNA) or predetermined mutant forms made bymutating the DNA, either to arrive at an allele or a variant not foundin nature. In general, the location and nature of the mutation chosenwill depend upon the rPTK characteristic to be modified. Obviously, suchvariations that, for example, convert rPTK into a known receptor proteintyrosine kinase are not included within the scope of this invention, norare any other rPTK variants or polypeptide sequences that are not noveland unobvious over the prior art.

[0104] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. Deletions may be introduced into regions of low homology amongrPTK and known rPTKs (which share the most sequence identity to thehuman rPTK amino acid sequence) to modify the activity of rPTK.Deletions from rPTK in areas of substantial homology with homologousrPTK proteins will be more likely to modify the biological activity ofrPTK more significantly. The number of consecutive deletions will beselected so as to preserve the tertiary structure of rPTK in theaffected domain, e.g., beta-pleated sheet or alpha helix.

[0105] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Intrasequence insertions (i.e., insertions within the mature rPTKsequence) may range generally from about 1 to 10 residues, morepreferably 1 to 5, most preferably 1 to 3. Insertions are preferablymade in even numbers of residues, but this is not required. Examples ofterminal insertions include mature rPTK with an N-terminal methionylresidue, an artifact of the direct expression of mature rPTK inrecombinant cell culture, and fusion of a heterologous N-terminal signalsequence to the N-terminus of the mature rPTK molecule to facilitate thesecretion of mature rPTK from recombinant hosts. Such signal sequencesgenerally will be obtained from, and thus homologous to, the intendedhost cell species. Suitable sequences include STII or lpp for E. coli,alpha factor for yeast, and viral signals such as herpes gD formammalian cells.

[0106] Other insertional variants of the rPTK molecule include thefusion to the N- or C-terminus of rPTK of immunogenic polypeptides,e.g., bacterial polypeptides such as beta-lactamase or an enzyme encodedby the E. coli trp locus, or yeast protein, and C-terminal fusions withproteins having a long half-life such as immunoglobulin constant regions(or other immunoglobulin regions), albumin, or ferritin, as described inWO 89/02922 published 6 Apr. 1989.

[0107] A third group of variants are amino acid substitution variants.These variants have at least one amino acid residue in the rPTK moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of rPTK and sites where the amino acidsfound in the known analogues are substantially different in terms ofside-chain bulk, charge, or hydrophobicity, but where there is also ahigh degree of sequence identity at the selected site within variousanimal rPTK species.

[0108] Other sites of interest are those in which particular residues ofthe rPTK obtained from various species are identical. These sites,especially those falling within a sequence of at least three otheridentically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of preferred substitutions. If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated exemplary substitutions in Table 1, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened. TABLE 1 Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser serGln (Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H) asn; gln; lys;arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L)norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala leu Pro (P) gly gly Ser(S) thr thr Thr (T) ser ser Trp (W) tyr tyr Tyr (Y) trp; phe; thr; serphe Val (V) ile; leu; met; phe; leu ala; norleucine

[0109] Substantial modifications in function or immunological identityof the rPTK are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0110] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0111] (2) neutral hydrophilic: cys, ser, thr;

[0112] (3) acidic: asp, glu;

[0113] (4) basic: asn, gln, his, lys, arg;

[0114] (5) residues that influence chain orientation: gly, pro; and

[0115] (6) aromatic: trp, tyr, phe.

[0116] Substantial modifications in enzymatic function are accomplishedby deletions, or replacement of, tyrosine residues in the catalyticdomain of the native rPTK as these modifications may well disrupt thetyrosine kinase activity of the receptor.

[0117] Non-conservative substitutions will entail exchanging a member ofone of these classes for another. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

[0118] In one embodiment of the invention, it is desirable to inactivateone or more protease cleavage sites that are present in the molecule.These sites are identified by inspection of the encoded amino acidsequence, in the case of trypsin, e.g., for an arginyl or lysinylresidue. When protease cleavage sites are identified, they are renderedinactive to proteolytic cleavage by substituting the targeted residuewith another residue, preferably a basic residue such as glutamine or ahydrophobic residue such as serine; by deleting the residue; or byinserting a prolyl residue immediately after the residue.

[0119] In another embodiment, any methionyl residues other than thestarting methionyl residue of the signal sequence, or any residuelocated within about three residues N- or C-terminal to each suchmethionyl residue, is substituted by another residue (preferably inaccord with Table 1) or deleted. Alternatively, about 1-3 residues areinserted adjacent to such sites.

[0120] Any cysteine residues not involved in maintaining the properconformation of rPTK also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

[0121] Nucleic acid molecules encoding amino acid sequence variants ofrPTK are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of rPTK.

[0122] Oligonucleotide-mediated mutagenesis is a preferred method forpreparing substitution, deletion, and insertion variants of rPTK DNA.This technique is well known in the art as described by Adelman et al.,DNA, 2: 183 (1983). Briefly, rPTK DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of rPTK. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that will thus incorporate theoligonucleotide primer, and will code for the selected alteration in therPTK DNA.

[0123] Generally, oligonucleotides of at least 25 nucleotides in lengthare used. An optimal oligonucleotide will have 12 to 15 nucleotides thatare completely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA, 75: 5765 (1978).

[0124] The DNA template can be generated by those vectors that areeither derived from bacteriophage M13 vectors (the commerciallyavailable M13 mp18 and M13 mp19 vectors are suitable), or those vectorsthat contain a single-stranded phage origin of replication as describedby Viera et al. Meth. Enzymol., 153: 3 (1987). Thus, the DNA that is tobe mutated may be inserted into one of these vectors to generatesingle-stranded template. Production of the single-stranded template isdescribed in Sections 4.21-4.41 of Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, NY 1989).

[0125] Alternatively, single-stranded DNA template may be generated bydenaturing double-stranded plasmid (or other) DNA using standardtechniques.

[0126] For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of rPTK, and the other strand (the original template) encodes thenative, unaltered sequence of rPTK. This heteroduplex molecule is thentransformed into a suitable host cell, usually a prokaryote such as E.coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with³²P to identify the bacterial colonies that contain the mutated DNA. Themutated region is then removed and placed in an appropriate vector forprotein production, generally an expression vector of the type typicallyemployed for transformation of an appropriate host.

[0127] The method described immediately above may be modified such thata homoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained from theAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(aS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion.

[0128] After the template strand of the double-stranded heteroduplex isnicked with an appropriate restriction enzyme, the template strand canbe digested with ExoIII nuclease or another appropriate nuclease pastthe region that contains the site(s) to be mutagenized. The reaction isthen stopped to leave a molecule that is only partially single-stranded.A complete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

[0129] DNA encoding rPTK mutants with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

[0130] In the first method, a separate oligonucleotide is generated foreach amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions.

[0131] The alternative method involvers two or more rounds ofmutagenesis to produce the desired mutant. The first round is asdescribed for the single mutants: wild-type DNA is used for thetemplate, an oligonucleotide encoding the first desired amino acidsubstitution(s) is annealed to this template, and the heteroduplex DNAmolecule is then generated. The second round of mutagenesis utilizes themutated DNA produced in the first round of mutagenesis as the template.Thus, this template already contains one or more mutations. Theoligonucleotide encoding the additional desired amino acidsubstitution(s) is then annealed to this template, and the resultingstrand of DNA now encodes mutations from both the first and secondrounds of mutagenesis. This resultant DNA can be used as a template in athird round of mutagenesis, and sc. on.

[0132] PCR mutagenesis is also suitable for making amino acid variantsof rPTK. While the following discussion refers to DNA, it is understoodthat the technique also finds application with RNA. The PCR techniquegenerally refers to the following procedure (see Erlich, supra, thechapter by R. Higuchi, p. 61-70): When small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer is located within 200 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

[0133] If the ratio of template to product material is extremely low,the vast majority of product DNA fragments incorporate the desiredmutation(s). This product material is used to replace the correspondingregion in the plasmid that served as PCR template using standard DNAtechnology. Mutations at separate positions can be introducedsimultaneously by either using a mutant second primer, or performing asecond PCR with different mutant primers and ligating the two resultingPCR fragments simultaneously to the vector fragment in a three (ormore)-part ligation.

[0134] In a specific example of PCR mutagenesis, template plasmid DNA (1μg) is linearized by digestion with a restriction endonuclease that hasa unique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp® kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlaid with 35 μl mineral oil. The reaction mixture is denaturedfor five minutes at 100° C., placed briefly on ice, and then 1 μlThermus aquaticus (Taq) DNA polymerase (5 units/μl, purchased fromPerkin-Elmer Cetus) is added below the mineral oil layer. The reactionmixture is then inserted into a DNA Thermal Cycler (purchased fromPerkin-Elmer Cetus) programmed as follows:

[0135] 2 min. 55° C.

[0136] 30 sec. 72° C., then 19 cycles of the following:

[0137] 30 sec. 94° C.

[0138] 30 sec. 55° C., and

[0139] 30 sec. 72° C.

[0140] At the end of the program, the reaction vial is removed from thethermal cycler and the aqueous phase transferred to a new vial,extracted with phenol/chloroform (50:50 vol), and ethanol precipitated,and the DNA is recovered by standard procedures. This material issubsequently subjected to the appropriate treatments for insertion intoa vector.

[0141] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al., Gene, 34: 315 (1985).The starting material is the plasmid (or other vector) comprising therPTK DNA to be mutated. The codon(s) in the rPTK DNA to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the rPTK DNA. After the restriction sites havebeen introduced into the plasmid, the plasmid is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedrPTK DNA sequence.

[0142] C. Insertion of Nucleic Acid into Replicable Vector

[0143] The nucleic acid (e.g., cDNA or genomic DNA) encoding native orvariant rPTK is inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable, and selection of the appropriate vector will depend on 1)whether it is to be used for DNA amplification or for DNA expression, 2)the size of the nucleic acid to be inserted into the vector, and 3) thehost cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and the host cell with which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

[0144] (i) Signal Sequence Component

[0145] The rPTKs of this invention may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. In general, the signal sequence may be a component ofthe vector, or it may be a part of the rPTK DNA that is inserted intothe vector. The heterologous signal sequence selected should be one thatis recognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe native rPTK signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182 issued 23 Apr. 1991), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published 4 Apr. 1990), or the signal described in WO 90/13646 published15 Nov. 1990. In mammalian cell expression the native signal sequence(i.e., the rPTK presequence that normally directs secretion of rPTK fromhuman cells in vivo) is satisfactory, although other mammalian signalsequences may be suitable, such as signal sequences from other animalrPTKs, and signal sequences from secreted polypeptides of the same orrelated species, as well as viral secretary leaders, for example, theherpes simplex gD signal.

[0146] The DNA for such precursor region is ligated in reading frame toDNA encoding the mature rPTK.

[0147] (ii) Origin of Replication Component

[0148] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0149] Most expression vectors are “shuttle” vectors, i.e., they arecapable of replication in at least one class of organisms but can betransfected into another organism for expression. For example, a vectoris cloned in E. coli and then the same vector is transfected into yeastor mammalian cells for expression even though it is not capable ofreplicating independently of the host cell chromosome.

[0150] DNA may also be amplified by insertion into the host genome.

[0151] This is readily accomplished using Bacillus species as hosts, forexample, by including in the vector a DNA sequence that is complementaryto a sequence found in Bacillus genomic DNA. Transfection of Bacilluswith this vector results in homologous recombination with the genome andinsertion of rPTK DNA. However, the recovery of genomic DNA encodingrPTK is more complex than that of an exogenously replicated vectorbecause restriction enzyme digestion is required to excise the rPTK DNA.

[0152] (iii) Selection Gene Component

[0153] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D alanine racemase for Bacilli.

[0154] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene express a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin (Southern et al., J. Molec. Appl.Genet., 1: 327 [1982]), mycophenolic acid (Mulligan et al., Science,209: 1422 [1980]) or hygromycin (Sugden et al., Mol. Cell. Biol., 5:410-413 [1985]). The three examples given above employ bacterial genesunder eukaryotic control to convey resistance to the appropriate drugG418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin,respectively.

[0155] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competent totake up the rPTK nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesrPTK. Amplification is the process by which genes in greater demand forthe production of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.

[0156] Increased quantities of rPTK are synthesized from the amplifiedDNA. Other examples of amplifiable genes include metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

[0157] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contains methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell when wild-type DHFR is employed is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77: 4216 (1980). The transformed cells are then exposedto increased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingrPTK. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060).

[0158] Alternatively, host cells [particularly wild-type hosts thatcontain endogenous DHFR] transformed or co-transformed with DNAsequences encoding rPTK, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′ phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0159] A suitable selection gene for use in yeast is the txp1 gene 39[1979]; Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al., Gene,10: 157 [1980]). The txp1 gene provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presenceof the trp1 lesion in the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC20,622 or 38,626) are complemented by known plasmids bearing the Leu2gene.

[0160] In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can be used for transformation of Kluyveromyces yeasts. Bianchi etal., Curr. Genet., 12: 185 (1987). More recertly, an expression systemfor large-scale production of recombinant calf chymosin was reported forK. lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Stablemulti-copy expression vectors for secretion of mature recombinant humanserum albumin by industrial strains of Kluyveromyces have also beendisclosed. Fleer et al., Bio/Technology, 9: 968-975 (1991).

[0161] (iv) Promoter Component

[0162] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the rPTKnucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of particularnucleic acid sequence, such as the rPTK nucleic acid sequence, to whichthey are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked torPTK-encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native rPTK promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the rPTK DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of rPTK as compared to the native rPTK promoter.

[0163] Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615t19781; and Goeddel et al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding rPTK (Siebenlist et al., Cell,20: 269 [1980]) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding rPTK.

[0164] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

[0165] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255: 2.073 [1980]) or other glycolytic enzymes (Hess etal., J. Adv. Enzyme Reg., 7: 149 [1968]; and Holland, Biochemistry, 17:4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0166] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

[0167] rPTK transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with therPTK sequence, provided such promoters are compatible with the host cellsystems.

[0168] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication. Fiers et al., Nature, 273:113 (1978);Mulligan and Berg, Science, 209: 1422-1427 (1980); Pavlakis et al.,Proc. Natl. Acad. Sci. USA, 78: 7398-7402 (1981). The immediate earlypromoter of the human cytomegalovirus is conveniently obtained as aHindIII E restriction fragment. Greenaway et al., Gene, 18: 355-360(1982). A system for expressing DNA in mammalian hosts using the bovinepapilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso Gray et al., Nature, 295: 503-508 (1982) on expressing cDNAencoding immune interferon in monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170(1982) on expression of the human interferon μ1 gene in cultured mouseand rabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

[0169] (v) Enhancer Element Component

[0170] Transcription of a DNA encoding the rPTK of this invention byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993[1981]) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108 [1983]) to thetranscription unit, within an intron (Banerji et al., Cell, 33: 729[1983]), as well as within the coding sequence itself (Osborne et al.,Mol. Cell Bio., 4: 1293 [1984]). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18(1-982) on enhancing elements for activation of eukaryotic promoters.The enhancer may be spliced into the vector at a position 5′ or 3′ tothe rPTK-encoding sequence, but is preferably located at a site 5′ fromthe promoter.

[0171] (vi) Transcription Termination Component

[0172] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding rPTK.

[0173] (vii) Construction and Analysis of Vectors

[0174] Construction of suitable vectors containing one or more of theabove listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

[0175] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate.

[0176] Plasmids from the transformants are prepared, analyzed byrestriction endonuclease digestion, and/or sequenced by the method ofMessing et al., Nucleic Acids Res., 9: 309 (1981) or by the LO method ofMaxam et al., Methods in Enzymology, 65: 499 (1980).

[0177] (viii) Transient Expression Vectors

[0178] Particularly useful in the practice of this invention areexpression vectors that provide for the transient expression inmammalian cells of DNA encoding rPTK. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17-16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptides encoded by cloned DNAS, as wellas for the rapid screening of such polypeptides for desired biologicalor physiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of rPTK that are biologically active rPTK.

[0179] (ix) Suitable Exemplary Vertebrate Cell Vectors

[0180] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of rPTK in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293: 620-625 (1981); Mantei et al.,Nature, 281: 40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058.A particularly useful plasmid for mammalian cell culture expression ofrPTK is pRK5 (EP pub. no. 307,247) or pSVI6B (PCT pub. no. WO 91/08291published 13 Jun. 1991).

[0181] D. Selection and Transformation of Host Cells

[0182] Suitable host cells for cloning or expressing the vectors hereinare the prokaryote, yeast, or higher eukaryote cells described above.Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 is aparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 27C7. The complete genotype of 27C7 is tonAΔ ptr3 phoAΔE15a(argF-lac)169 ompTΔ degP41kan^(r). Strain 27C7 was deposited on 30 Oct.1991 in the American Type Culture Collection as ATCC No. 55,244.Alternatively, the strain of E. coli having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 may be employed.Alternatively, methods of cloning, e.g., PCR or other nucleic acidpolymerase reactions, are suitable.

[0183] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forrPTK-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 publishedMay 2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer etal., supra) such as, e.g., K. lactis [MW98-8C, CBS683, CBS4574;Louvencourt et al., J. Bacteriol., 737 (1983)], K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg etal., supra), K. thermotolerans, and K. znarxianus; yarrowia [EP402,226]; Pichia pastoris (EP 183,070; Sreekrishna et al., J. BasicMicrobiol., 28: 265-278 [1988]); Candida; Trichoderma reesia [EP244,234]; Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76: 5259-5263 [1979]); Schwanniomyces such as Schwanniomycesoccidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungisuch as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans(Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983];Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl.Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes,EMBO J., 4: 475-479 [1985]).

[0184] Suitable host cells for the expression of glycosylated rPTK arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6: 47-55(1988); Miller et al., in Genetic Engineering, Setlow, J. K. et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature, 315: 592-594 (1985). A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

[0185] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the rPTK DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding the rPTK is transferred to the plantcell host such that it is transfected, and will, under appropriateconditions, express the rPTK DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published 21 Jun. 1989.

[0186] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, Academic Press, Kruseand Patterson, editors [1973]). Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36: 59[1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77: 4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

[0187] Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

[0188] Transfection refers to the taking up of an expression vector by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

[0189] Transformation means introducing DNA into an organism so that theDNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in section 1.82 ofSambrook et al., supra, or electroporation is generally used forprokaryotes or other cells that contain substantial cell-wall barriers.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23: 315 (1983)and WO 89/05859 published 29 Jun. 1989. In addition, plants may betransfected using ultrasound treatment as described in WO 91/00358published 10 Jan. 1991.

[0190] For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) is preferred. General aspects of mammalian cell hostsystem transformations have been described by Axel in U.S. Pat. No.4,399,216 issued 16 Aug. 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829(1979). However, other methods for introducing DNA into cells, such asby nuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology (1989), Keown et al.,Methods in Enzymology, 185: 527-537 (1990), and Mansour et al., Nature,336: 348-352 (1988).

[0191] E. Culturing the Host Cells

[0192] Prokaryotic cells used to produce the rPTK polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

[0193] The mammalian host cells used to produce the rPTK of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium([DMEM], Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz., 58: 44(1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of allof which are incorporated herein by reference, may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source: Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

[0194] In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed., IRLPress, 1991.

[0195] The host cells referred to in this disclosure encompass cells inculture as well as cells that are within a host animal.

[0196] F. Detecting Gene Amplification/Expression

[0197] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³²P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

[0198] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

[0199] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native rPTK polypeptide or against a synthetic peptide basedon the DNA sequences provided herein as described further in Section 3below.

[0200] G. Purification of rPTK Polypeptide

[0201] rPTK preferably is recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysates when directly expressed without a secretory signal.

[0202] When rPTK is expressed in a recombinant cell other than one ofhuman origin, the rPTK is completely free of proteins or polypeptides ofhuman origin. However, it is necessary to purify rPTK from recombinantcell proteins or polypeptides to obtain preparations that aresubstantially homogeneous as to rPTK. As a first step, the culturemedium or lysate is centrifuged to remove particulate cell debris. rPTKthereafter is purified from contaminant soluble proteins andpolypeptides, with the following procedures being exemplary of suitablepurification procedures: by fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to, remove contaminants such as IgG.

[0203] rPTK variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion as native rPTK, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a rPTK fusion with anotherprotein or polypeptide, e.g., a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion LS polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-rPTK column can be employed toabsorb the rPTK variant by binding it to at least one remaining immuneepitope. A protease inhibitor such as phenyl methyl sulfonyl fluoride(PMSF) also may be useful to inhibit proteolytic degradation duringpurification, and antibiotics may be included to prevent the growth ofadventitious contaminants. One skilled in the art will appreciate thatpurification methods suitable for native rPTK may require modificationto account for changes in the character of rPTK or its variants uponexpression in recombinant cell culture.

[0204] H. Covalent Modifications of rPTK Polypeptides

[0205] Covalent modifications of rPTK polypeptides are included withinthe scope of this invention. Both native rPTK and amino acid sequencevariants of the rPTK may be covalently modified. One type of covalentmodification included within the scope of this invention is a rPTKfragment. Variant rPTK fragments having up to about 40 amino acidresidues may be conveniently prepared by chemical synthesis or byenzymatic or chemical cleavage of the full-length or variant rPTKpolypeptide. Other types of covalent modifications of the rPTK orfragments thereof are introduced into the molecule by reacting targetedamino acid residues of the rPTK or fragments thereof with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

[0206] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl) propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0207] Histidyl residues are derivatized by reaction withdiethyl-pyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

[0208] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrob nzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0209] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0210] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

[0211] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R—N═C═N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

[0212] Derivatization with bifunctional agents is useful forcrosslinking rPTK to a water-insoluble support matrix or surface for usein the method for purifying anti-rPTK antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0213] U.S. Pat. No. 4,330,440 are employed for protein immobilization.

[0214] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

[0215] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0216] Another type of covalent modification of the rPTK polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. By altering is meantdeleting one or more carbohydrate moieties found in native rPTK, and/oradding one or more glycosylation sites that are not present in thenative rPTK.

[0217] Glycosylation of polypeptides is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0218] Addition of glycosylation sites to the rPTK polypeptide isconveniently accomplished by altering the amino acid sequence suchsequences (for N-linked glycosylation sites). The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the native rPTK sequence (for O-linkedglycosylation sites). For ease, the rPTK amino acid sequence ispreferably altered through changes at the DNA level, particularly bymutating the DNA encoding the rPTK polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids. The DNA mutation(s) may be made using methods described aboveunder the heading of “Amino Acid Sequence Variants of rPTK Polypeptide.”

[0219] Another means of increasing the number of carbohydrate moietieson the rPTK polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. These procedures are advantageous in thatthey do not require production of the LS polypeptide in a host cell thathas glycosylation capabilities for N- or O-linked-glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published 11Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981).

[0220] Removal of carbohydrate moieties present on the rPTK polypeptidemay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al.,Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydratemoieties on polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al., Meth.Enzmmol., 138: 350 (1987).

[0221] Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al., J.Biol. Chem., 257: 3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

[0222] Another type of covalent modification of rPTK comprises linkingthe rPTK polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

[0223] rPTK also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate]microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A., Ed., (1980).

[0224] rPTK preparations are also useful in generating antibodies, asstandards in assays for rPTK (e.g., by labeling rPTK for use as astandard in a radioimmunoassay, enzyme-linked immunoassay, orradioreceptor assay), in affinity purification techniques, and incompetitive-type receptor binding assays when labeled with radioiodine,enzymes, fluorophores, spin labels, and the like.

[0225] Since it is often difficult to predict in advance thecharacteristics of a variant rPTK, it will be appreciated that somescreening of the recovered variant will be needed to select the optimalvariant. For example, one can screen for protein kinase activity usingthe techniques set forth in Lokker et al., EMBO, 11, 2503-2510 (1992). Achange in the immunological character of the rPTK molecule, such asaffinity for a given antibody, is also able to be measured by acompetitive-type immunoassay. The variant is assayed for changes in thesuppression or enhancement of its enzymatic activity by comparison tothe activity observed for native RPTK in the same assay. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

[0226] 2. Uses. Therapeutic Compositions and Administration of rPTK

[0227] rPTK is believed to find therapeutic use for treating mammals viastimulation of cell growth and/or differentiation. For example, Rse orHPTK6 may be used to treat neuro-degenerative diseases (e.g. seniledementia of the Alzheimer's type, peripheral neuropathies, Parkinson'sdisease and Huntington's disease) or diseases of the kidney (e.g.,glomerulus sclerosis, which is associated with diabetes). Rse maysimilarly be used to generate the production of platelets frommegakaryocytes. Hence, the Rse may find utility for use in relation tobone marrow transplants, for example.

[0228] The nucleic acid encoding the rPTK may be used as a diagnosticfor tissue-specific typing. For example, such procedures as in situhybridization, Northern and Southern blotting, and PCR analysis may beused to determine whether DNA and/or RNA encoding rPTK is present in thecell type(s) being evaluated.

[0229] Isolated rPTK polypeptide may also be used in quantitativediagnostic assays as a standard or control against which samplescontaining unknown quantities of rPTK may be prepared.

[0230] Therapeutic formulations of rPTK for treating neuro-degenerativeor kidney diseases are prepared for storage by mixing rPTK having thedesired degree of purity with optional physiologically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, supra), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

[0231] rPTK to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. rPTKordinarily will be stored in lyophilized form or in solution.

[0232] Therapeutic rPTK compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0233] The route of rPTK, or rPTK antibody administration is in accordwith known methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained release systemsas noted below. rPTK is administered continuously by infusion or bybolus injection. rPTK antibody is administered in the same fashion, orby administration into the blood stream or lymph.

[0234] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981)and Langer, Chem. Tech., 12: 98-105 (1982) or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

[0235] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0236] Sustained-release rPTK compositions also include liposomallyentrapped rPTK. Liposomes containing rPTK are prepared by methods knownper se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal rPTK therapy.

[0237] An effective amount of rPTK to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer rPTK until a dosage is reached that achievesthe desired effect. The progress of this therapy is easily monitored byconventional assays.

[0238] 3. rPTK Antibody Preparation

[0239] The antibodies of this invention are obtained by routinescreening. Polyclonal antibodies to the rPTK generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the rPTK and an adjuvant. It may be useful to conjugate the rPTK or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

[0240] The route and schedule of the host animal or culturedantibody-producing cells therefrom are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction. While mice are frequently employed as the test model, it iscontemplated that any mammalian subject including human subjects orantibody-producing cells obtained therefrom can be manipulated accordingto the processes of this invention to serve as the basis for productionof mammalian, including human, hybrid cell lines.

[0241] Animals are typically immunized against the immunogenicconjugates or derivatives by combining 1 mg or 1 μg of conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later the animals are boosted with ⅕ to {fraction (1/10)} theoriginal amount of conjugate in Freund's complete adjuvant (or othersuitable adjuvant) by subcutaneous injection at multiple sites. 7 to 14days later animals are bled and the serum is assayed for anti-rPTKtiter. Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same rPTK, but conjugated toa different protein and/or through a different cross-linking agent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are used to enhance theimmune response.

[0242] After immunization, monoclonal antibodies are prepared byrecovering immune cells (typically spleen cells or lymphocytes fromlymph node tissue) from immunized animals and immortalizing the cells inconventional fashion, e.g., by fusion with myeloma cells or byEpstein-Barr (EB)-virus transformation and screening for clonesexpressing the desired antibody. The hybridoma technique describedoriginally by Kohler and Milstein, Eur. J. Immunol., 6: 511 (1976), andalso described by Hammerling et al., In: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981) has been widelyapplied to produce hybrid cell lines that secrete high levels ofmonoclonal antibodies against many specific antigens.

[0243] It is possible to fuse cells of one species with another.However, it is preferable that the source of the immunized antibodyproducing cells and the myeloma be from the same species.

[0244] The hybrid cell lines can be maintained in culture in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin-thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody.

[0245] The secreted antibody is recovered from tissue culturesupernatant by conventional methods such as precipitation, ion exchangechromatography, affinity chromatography, or the like. The antibodiesdescribed herein are also recovered from hybridoma cell cultures byconventional methods for purification of IgG or IgM, as the case may be,that heretofore have been used to purify these immunoglobulins frompooled plasma, e.g., ethanol or polyethylene glycol precipitationprocedures. The purified antibodies are sterile filtered, and optionallyare conjugated to a detectable marker such as an enzyme or spin labelfor use in diagnostic assays of the rPTK in test samples.

[0246] While routinely mouse monoclonal antibodies are used, theinvention is not so limited; in fact, human antibodies may be used andmay prove to be preferable. Such antibodies can be obtained by usinghuman hybridomas (Cote et al., Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, p. 77 [1985]). In fact, according to the invention,techniques developed for the production of chimeric antibodies (Morrisonet al., Proc. Natl. Acad. Sci., 81: 6851 [1984]); Neuberger et al.,Nature, 312: 604 [1984]; Takeda et al., Nature, 314: 452 [1985]; EP184,187; EP 171,496; EP 173,494; PCT WO 86/01533; Shaw et al., J. Nat.Canc. Inst., 80: 1553-1559 [1988]; Morrison, Science, 229: 1202-1207[1985]; and Oi et al., BioTechniques, 4: 214 [1986]) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity (such as ability to activate human complement andmediate ADCC) can be used; such antibodies are within the scope of thisinvention.

[0247] In a preferred embodiment of the invention, humanized antibodiesare used to reduce or eliminate any anti-globulin immune response inhumans. As used herein, the term “humanized” antibody is an embodimentof chimeric antibodies wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some amino acid residues from thecomplementarity determining regions (CDRs), the hypervariable regions inthe variable domains which are directly involved with formation of theantigen-binding site, and possibly some amino acids from the frameworkregions (FRs), the regions of sequence that are somewhat conservedwithin the variable domains, are substituted by residues from analogoussites in rodent antibodies. The construction of humanized antibodies isdescribed in Riechmann et al., Nature, 332: 323-327 (1988), Queen etal., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989), Co et al.,Proc. Natl. Acad. Sci. USA, 88: 2869-2873 (1991), Gorman et al., Proc.Natl. Acad. Sci., 88: 4181-4185 (1991), Daugherty et al., Nucleic AcidsRes., 19: 2471-2476 (1991), Brown et al., Proc. Natl. Acad. Sci. USA,88: 2663-2667 (1991), Junghans et al., Cancer Res., 50: 1495-1502(1990), Fendly et al., Cancer Res., 50: 1550-1558 (1990) and in PCTapplication WO 89/06692.

[0248] In some cases, substituting CDRs from rodent antibodies for thehuman CDRs in human frameworks is sufficient to transfer high antigenbinding affinity (Jones et al., Nature, 321: 522-525 [1986]; Verhoeyenet al., Science, 239: 1534-1536 [1988]) whereas in other cases it isnecessary to additionally replace one (Riechmann et al., supra) orseveral (Queen et al., supra) FR residues. See also Co et al., supra.

[0249] In a particularly preferred embodiment of the invention, thehumanized antibodies are designed and constructed according to themethods described in PCT application WO 92/22653, the entire disclosureof which is specifically incorporated herein by reference.

[0250] Techniques for creating recombinant DNA versions of theantigen-binding regions of antibody molecules (known as Fab fragments),which bypass the generation of monoclonal antibodies, are encompassedwithin the practice of this invention. One extracts antibody-specificmessenger RNA molecules from immune system cells taken from an immunizedanimal, transcribes these into complementary DNA (cDNA), and clones thecDNA into a bacterial expression system. One example of such a techniquesuitable for the practice of this invention was developed by researchersat Scripps/Stratagene, and incorporates a proprietary bacteriophagelambda vector system that contains a leader sequence that causes theexpressed Fab protein to migrate to the periplasmic space (between thebacterial cell membrane and the cell wall) or to be secreted. One canrapidly generate and screen great numbers of functional Fab fragmentsfor those that bind the antigen. Such rPTK-binding molecules (Fabfragments with specificity for the rPTK) are specifically encompassedwithin the term “antibody” as it is defined, discussed, and claimedherein.

[0251] The antibody preferably does not cross-react with other knownreceptor protein tyrosine kinases.

[0252] 4. Uses of rPTK Antibodies

[0253] rPTK antibodies may be used as ligands to the rPTK and are alsouseful in diagnostic assays for rPTK, e.g., detecting its expression inspecific cells, tissues, or serum. The antibodies are labeled in thesame fashion as rPTK described above and/or are immobilized on aninsoluble matrix. In one embodiment of a receptor binding assay, anantibody composition that binds to all or a selected plurality ofmembers of the rPTK family is immobilized on an insoluble matrix, thetest sample is contacted with the immobilized antibody composition toadsorb all rPTK family members, and then the immobilized family membersare contacted with a plurality of antibodies specific for each member,each of the antibodies being individually identifiable as specific for apredetermined family member, as by unique labels such as discretefluorophores or the like. By determining the presence and/or amount ofeach unique label, the relative proportion and amount of each familymember can be determined.

[0254] The antibodies of this invention are also useful in passivelyimmunizing patients.

[0255] rPTK antibodies also are useful for the affinity purification ofrPTK or rPTK ECD from recombinant cell culture or natural sources. rPTKantibodies that do not detectably cross-react with other receptorprotein tyrosine kinases can be used to purify rPTK or rPTK ECD freefrom these other known proteins.

[0256] Suitable diagnostic assays for rPTK and its antibodies are wellknown per se. For example, competitive, sandwich and steric inhibitionimmunoassay techniques are useful. The competitive and sandwich methodsemploy a phase-separation step as an integral part of the method whilesteric inhibition assays are conducted in a single reaction mixture.Fundamentally, the same procedures are used for the assay of rPTK andfor substances that bind rPTK, although certain methods will be favoreddepending upon the molecular weight of the substance being assayed.Therefore, the substance to be tested is referred to herein as ananalyte, irrespective of its status otherwise as an antigen or antibody,and proteins that bind to the analyte are denominated binding partners,whether they be antibodies, cell surface receptors, or antigens.

[0257] Analytical methods for rPTK or its antibodies all use one or moreof the following reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner, andsteric conjugates. The labeled reagents also are known as “tracers.”

[0258] The label used (and this is also useful to label rPTK nucleicacid for use as a probe) is any detectable functionality that does notinterfere with the binding of analyte and its binding partner. Numerouslabels are known for use in immunoassay, examples including moietiesthat may be detected directly, such as fluorochrome, chemiluminscent,and radioactive labels, as well as moieties, such as enzymes, that mustbe reacted or derivatized to be detected. Examples of such labelsinclude the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophoressuch as rare earth chelates or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g.,firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase,1-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and xanthineoxidase, coupled with an enzyme that employs hydrogen peroxide tooxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

[0259] Those of ordinary skill in the art will know of other suitablelabels that may be employed in accordance with the present invention.The binding of these labels to rPTK, antibodies, or fragments thereofcan be accomplished using standard techniques commonly known to those ofordinary skill in the art. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the polypeptide with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014,1021 (1974); Pain et al., J. Immunol.Methods, 40: 219-230 (1981); Nygren, J. Histochem. and Cytochem., 30:407-412 (1982); O'Sullivan et al., Methods in Enzymology, ed. J. J.Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y.,1981), pp. 147-166; Kennedy et al., Clin. Chim. Acta, 70: 1-31 (1976);and Schurs et al., Clin. Chim. Acta, 81: 1-40 (1977) coupling techniquesmentioned in the lattermost reference are the glutaraldehyde method, theperiodate method, the dimaleimide method, and them-maleimidobenzyl-N-hydroxysuccinimide ester method.

[0260] In the practice of the present invention, enzyme labels are apreferred embodiment. No single enzyme is ideal for use as a label inevery conceivable assay. Instead, one must determine which enzyme issuitable for a particular assay system. Criteria important for thechoice of enzymes are turnover number of the pure enzyme (the number ofsubstrate molecules converted to product per enzyme site per unit oftime), purity of the enzyme preparation, sensitivity of detection of itsproduct, ease and speed of detection of the enzyme reaction, absence ofinterfering factors or of enzyme-like activity in the test fluid,stability of the enzyme and its conjugate, availability and cost of theenzyme and its conjugate, and the like. Included among the enzymes usedas preferred labels in the assays of the present invention are alkalinephosphatase, HRP, beta-galactosidase, urease, glucose oxidase,glucoamylase, malate dehydrogenase, and glucose-6-phosphatedehydrogenase. Urease is among the more preferred enzyme labels,particularly because of chromogenic pH indicators that make its activityreadily visible to the naked eye.

[0261] Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

[0262] Other assay methods, known as competitive or sandwich assays, arewell established and widely used in the commercial diagnostics industry.

[0263] Competitive assays rely on the ability of a tracer analogue tocompete with the test sample analyte for a limited number of bindingsites on a common binding partner. The binding partner generally isinsolubilized before or after the competition and then the tracer andanalyte bound to the binding partner are separated from the unboundtracer and analyte. This separation is accomplished by decanting (wherethe binding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample analyte is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of analyte are prepared andcompared with the test results to quantitatively determine the amount ofanalyte present in the test sample. These assays are called ELISAsystems when enzymes are used as the detectable markers.

[0264] Another species of competitive assay, called a “homogeneous”assay, does not require a phase separation. Here, a conjugate of anenzyme with the analyte is prepared and used such that when anti-analytebinds to the analyte the presence of the anti-analyte modifies theenzyme activity. In this case, rPTK or its immunologically activefragments are conjugated with a bifunctional organic bridge to an enzymesuch as peroxidase. Conjugates are selected for use with anti-rPTK sothat binding of the anti-rPTK inhibits or potentiates the enzymeactivity of the label. This method per se is widely practiced under thename of EMIT.

[0265] Steric conjugates are used in steric hindrance methods forhomogeneous assay. These conjugates are synthesized by covalentlylinking a low-molecular-weight hapten to a small analyte so thatantibody to hapten substantially is unable to bind the conjugate at thesame time as anti-analyte. Under this assay procedure the analytepresent in the test sample will bind anti-analyte, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

[0266] Sandwich assays particularly are useful for the determination ofrPTK or rPTK antibodies. In sequential sandwich assays an immobilizedbinding partner is used to adsorb test sample analyte, the test sampleis removed as by washing, the bound analyte is used to adsorb labeledbinding partner, and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample analyte. In “simultaneous” sandwich assays the test sample is notseparated before adding the labeled binding partner. A sequentialsandwich assay using an anti-rPTK monoclonal antibody as one antibodyand a polyclonal anti-rPTK antibody as the other is useful in testingsamples for rPTK activity.

[0267] The foregoing are merely exemplary diagnostic assays for rPTK andantibodies. Other methods now or hereafter developed for thedetermination of these analytes are included within the scope hereof,including the bioassays described above.

[0268] 5. rPTK Ligand Preparation

[0269] As discussed above, rPTK ligands can comprise antibodies(including polyclonal antibodies, monoclonal antibodies and humanizedmonoclonal antibodies) against the rPTK. Other protein and non-proteinligands are also contemplated within the scope of the invention.

[0270] The ligand preferably constitutes the endogenous ligand to therPTK. In order to isolate the endogenous rPTK ligand, primary cellspurified from natural sources (e.g., blood tissue extracts or urine) orcell lines expressing the ligands are screened for the ligand. Cellsused to isolate the ligands may, for example, be selected from humankidney and brain cells. Cell lines can be established using well knowntechniques such as immortalization of the cells via transformation withviral DNA (e.g., SV40 DNA).

[0271] The endogenous ligand can then be identified and isolated usingtechniques which have been established in the art. For example, thetechniques disclosed in WO/92/20798 can be used to isolate the ligand tothe rPTK. Generally, the ligand will be recovered from a cellularmembrane fraction or a secreted form of the ligand will be isolated fromthe culture medium. Accordingly, the culture medium or lysate iscentrifuged to remove particulate cell debris. The ligand is thenpurified from the soluble protein fraction or the membrane fraction ofthe culture lysate by biochemical separation. The following proceduresare exemplary of suitable purification procedures fractionation on animmunoaffinity or ion-exchange column; ethanol precipitation; reversedphase HPLC; chromatography on silica, Heparin Sepharose or on a cationexchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; and gel filtration using, for example, SephadexG-75. Each of the fractions can then be assayed for its ability tophosphorylate the rPTK (see Example 1 for a suitable assay for tyrosinekinase activity), in order to isolate the fraction containing the ligandto the rPTK. Further purification of the fraction can then be carriedout as required.

[0272] Alternatively, the techniques used by Flanagan et al., Cell, 63:185-194 (1990) can be carried out. Flanagan et al. isolated the ligandto the c-kit proto-oncogene by genetically fusing the c-kit ECI) toplacental alkaline phosphatase thereby forming a soluble receptoraffinity reagent with an enzyme tag that could be readily traced.Binding of the fusion proteins is detectable by the enzymatic activityof the alkaline phosphatase secreted into the medium. The fusion proteinso formed, termed APtag-KIT, binds with high affinity to cell linesexpressing the ligand of interest. The bound cells are then isolatedfrom the APtag-KIT complex.

[0273] Accordingly, a chimeric nucleic acid construct encoding the ECDof Rse or the ECD of HPTK6 fused to the secretable alkaline phosphatasemarker can be generated.

[0274] To clone the cDNA that encodes the ligand, a cDNA library isconstructed from the isolated cells in a suitable expression vector,such as the vectors discussed earlier herein. The library is thentransfected into host cells (see above) and cells having the ligand ontheir surface are detected using the techniques of Flanagan et al.Single cell suspensions are incubated with the APtag-KIT and, afterremoving APtag-KIT proteins which are not bound to the cells bycentrifugation, cells are panned on plates coated with antibodiesagainst alkaline phosphatase (Seed et al., Proc. Natl. Acad. Sci., 84:,3365-69 [1987]). Cells to which the antibodies are bound are isolatedand the DNA is extracted therefrom using techniques available to theskilled artisan.

[0275] 6. Uses. Therapeutic Compositions and Administration of rPTKLigand

[0276] rPTK ligands are believed to find therapeutic use for treatingmammals via stimulation of cell growth and/or differentiation. Forexample, Rse ligand may be used to treat neuro-degenerative diseases(e.g. senile dementia of the Alzheimer's type, peripheral neuropathies,Parkinson's disease and Huntington's disease) or diseases of the kidney(e.g., glomerulus sclerosis, which is associated with diabetes). Rseligand may also be used to generate the production of platelets frommegakaryocytes. Like Rse ligand, HPTK6 ligand may be used to treatkidney diseases, such as glomerulus sclerosis. An antagonist ligand forHPTK6 may find therapeutic use in the treatment of cancer, e.g. breastcancer.

[0277] Therapeutic formulations of rPTK ligand are prepared for storageby mixing the ligand having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, supra), in the form of lyophilizedcake or aqueous solutions.

[0278] rPTK ligand to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. rPTK ligand ordinarily will be stored in lyophilizedform or in solution.

[0279] Therapeutic rPTK ligand compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a LS hypodermicinjection needle.

[0280] The route of rPTK ligand administration is in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems as noted below.rPTK ligand is administered continuously by infusion or by bolusinjection.

[0281] An effective amount of rPTK ligand to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer rPTK ligand until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

[0282] 7. Uses. Therapeutic Compositions and Administration of rPTK ECD

[0283] As discussed above, rPTK ECD can be used for the identificationand isolation of ligands to the rPTKs using the techniques disclosed inFlanagan et al., supra, for example.

[0284] rPTK ECD is also believed to find use as a therapeutic compoundfor removal of excess systemic or tissue-localized rPTK ligand which hasbeen administered to a patient. Removal of excess ligand is particularlydesirably where the ligand may be toxic to the patient. The rPTK ECDacts to bind the ligand in competition with endogenous rPTKs in thepatient. Similarly, it is contemplated that the rPTK ECD can beadministered to a patient simultaneously, or subsequent to,administration of the ligand in the form of a sustained releasecomposition. The ECD acts as a soluble binding protein for the ligand,thereby extending the half-life of the ligand. Also, the ECD mayconstitute a ligand to the receptor in so far as it is able to bind to,and activate, the ECD of an adjacent membrane bound rPTK. Accordingly,the ECD may be used as a ligand to the rPTK.

[0285] The nucleic acid encoding the rPTK ECD may be used as adiagnostic for tissue-specific typing. For example, such procedures asin situ hybridization, Northern and Southern blotting, and PCR analysismay be used to determine whether DNA and/or RNA encoding rPTK is presentin the cell type(s) being evaluated.

[0286] Therapeutic formulations of rPTK ECD are prepared for storage bymixing rPTK ECD having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, supra), in the form of lyophilizedcake or aqueous solutions.

[0287] rPTK ECD to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. rPTKECD ordinarily will be stored in lyophilized form or in solution.

[0288] Therapeutic rPTK ECD compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0289] The route of rPTK ECD administration is in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems as noted below.rPTK ECD is administered continuously by infusion or by bolus injection.

[0290] An effective amount of rPTK ECD to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer rPTK ECD until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

[0291] The following examples are offered by way of illustration and notby way of limitation. The disclosures of all literature references citedin the specification are expressly incorporated herein by reference.

EXAMPLE 1 Isolation and Characterization of Rse

[0292] A. cDNA Cloning and Sequencing

[0293] Degenerate oligodeoxyribonucleotide primers were designed tosequences encoding conserved amino acids in tyrosine kinases (Lai etal., supra). These primers were used to amplify fragments of tyrosinekinase containing genes from cDNA prepared from human brain RNA.Amplified fragments were cloned and sequenced. Nestedoligodeoxyribonucleotide primers (pair A:5′-CGGATCCAC(AC)G(ATGC)GA(CT)(CT)T (SEQ ID NO: 13) and5′-GGAATTCC(TC)TC(AT)GGAG(CT)(AG)TCCA(TC)(TC)T (SEQ ID NO: 14); pair B:5′-CGGATCCATCCACAGAGATGT (SEQ ID NO: 15) and5′-GGAATTCCAAAGGACCA(GC)AC(GA)TC) (SEQ ID NO: 16) were used to amplifyfragments of cDNA prepared from human brain RNA. Amplified DNA fragmentswere cloned as BamHI and EcoRI inserts in pUC19 (see Hanks et al.,supra). Amplification reactions were performed using Taq DNA polymerasein a Perkin-Elmer 480 thermocycler, 40 cycles of 94° C. for 30 seconds,45° C. for 30 seconds, and 72° C. for 1 minute; primer-pair B was addedfollowing cycle 20. Recombinants were identified and sequenced using thedideoxynucleotide method. A 50 base single-strandedoligodeoxyribonucleotide probe(5′-GACCGTGTGTGTGGCTGACTTTGGACTCTCCTGGAAGATC (SEQ ID NO: 17)) was usedas a probe to screen 1.2×10⁶ plaques from a random-primed lambda gt10library prepared from. RNA isolated from human fetal brain. Conditionsfor plating libraries, hybridizing and washing filters were aspreviously described (Godowski, et al., Proc. Natl. Acad. Sci. 86:8083-8087 [1989]). One positive plaque was obtained, with an insert sizeof approximately 1.2 Kb. An oligodeoxyribonucleotideprobe(5′-GGCTGTGCCTCCAAATTGCCCGTCAAGTGGCTGGCCCTGG (SEQ ID NO: 18)) based onsequence obtained from the 5′ end of the 1.2 Kb clone was used to screen1.2×10⁶ plaques from an oligo dT-primed lambda gt10 library preparedfrom RNA from the Hep 3B cell line. The inserts from 15 positive plaqueswere characterized, and the largest insert, approximately 3.5 Kb inlength, was sequenced. An oligodeoxyribonucleotide primer(5′-AGCCGGTGAAGCTGAACTGCAGTGTGGAGGGGATGGAGGAGCCTGACATC (SEQ ID NO: 19))based on sequence from the 5 region of the 3.5 Kb clone was used toscreen 1.2×10⁶plaques from a second lambda gt10 Hep 3B library. Fourclones were obtained, and one of these contained a 3.0 Kb insert thatcontained the putative initiator methionine.

[0294] The murine homologue of Rse was obtained by screening a murinebrain cDNA library prepared in lambda gt10 (Clontech, Palo Alto Calif.)with a random-primed probe corresponding to nucleotides 1-1163 from thehuman Rse cDNA (FIG. 1A). Thirteen clones were purified and the size ofthe inserts was determined. Two overlapping clones, mbptk3.1 andmbptk3.8 (corresponding to nucleotides 737-3759 and 367-3785 of themurine Rse cDNA, respectively, of FIG. 1B) were sequenced. To obtain the5′ region of the murine Rse cDNA, an oligonucleotide probe derived fromthe 5′ end of the mbptk3.8 clone(5′-TCCAGCTACAACGCTAGCGTGGCCTGGGTGCCAGGTGCTGACGGCCTAGC (SEQ ID NO: 20))was used to rescreen the murine brain cDNA library. Two positive plaqueswere purified, and the 5′ end of the mbptk3.14 insert was sequenced andshown to contain the 5′ end of the the murine Rse cDNA.

[0295] The assembled nucleotide and deduced amino acid sequences ofhuman Rse are shown in FIG. 1A. The Rse cDNA sequence contains an openreading frame of 890 amino acids with two in-frame potential initiationcodons (Kozak, M., J. Cell Biol. 115: 887-903 [1991]). The first ofthese methionine codons precedes a hydrophobic region encoding aputative signal sequence of 40 amino acids (FIGS. 1A and 4). A secondhydrophobic region is located between amino acids 429-451 and may serveas a transmembrane domain (FIG. 4). This putative transmembrane regionis followed by 5 basic amino acids that are characteristic of a stoptransfer sequence. Thus, the mature form of human Rse is predicted tocontain an ECD of 388 amino acids and an ICD of 439 amino acids. Thehuman Rse cDNA was used as a basis to obtain overlapping clones encodingmurine Rse cDNA from a murine brain cDNA library. The assemblednucleotide and deduced amino acid sequences are shown in FIG. 1B. Themurine Rse cDNA sequence contains an open reading frame of 880 aminoacids. Murine Rse contains a potential signal sequence of 30 aminoacids, and a hydrophobic region between amino acids 419 and 441 that mayencode a transmembrane domain (FIGS. 1B and 4). The overall amino acidsequence identity of murine and human Rse is 90%, with a sequenceidentity of 85% in the ECD and 93% in the ICD. Human and murine Rsecontain significant homology in the ICD with a number of proteins. Aminoacids 650-703 of murine Rse matched the partial rat Tyro-3 sequence in54 out of 54 positions (Lai et al., supra); human Rse contains a singleamino acid difference with rat Tyro-3; Q⁷¹² of human Rse is replacedwith H in the rat sequence. Tyro-3 expression was detected at highlevels in the rat brain, and in several other tissues that wereexamined. In situ hybridization studies show that Tyro-3 is expressed ina highly restricted pattern within the brain, with strong hybridizationseen in the CA1 field but little hybridization observed in the CA2, CA3or CA4 fields of the hippocampus (Lai et al., supra)

[0296] The expression of Rse in murine brain samples was also analyzed,using a probe from the ECD portion of the murine cDNA to reduce thepossibility of cross-hybridization with mRNAs encoding other proteintyrosine kinases. An identical pattern of hybridization for murine Rsein the hippocampus as that previously reported for Tyro-3 was detected.

[0297] Taken together, these results indicate that Tyro-3 encodes aportion of the rat homologue of Rse. In the tyrosine kinase domain,human Rse was most similar to the human rPTKs Axl (64%), hepatocytegrowth factor (HGF) receptor (45%), insulin receptor (43%), insulin-likegrowth factor I (IGF-I) receptor (42%) and Ros (42%) [O'Bryan, J. P.,Mol. Cell. Biol. 11: 5016-5031 (1991); Janssen, J. W. G., et al.,Oncogene 6:2113-2120 (1991); Park M., et al., Proc. Natl. Acad. Sci. 84:6379-6383 (1987); Ullrich, A., et al., Nature 313: 756-761 (1985);Ullrich, A., et al., EMBO J. 5: 2503-2512 (1986); and Birchmeier, C., etal., Mol. Cell. Biol. 6: 3109-3116 (1986)]. Human and murine Rse containa consensus site for Mg²⁺-ATP binding (GxGxxG(x)₁₅₋₂₀,xKxM) beginning atamino acids 525 and 515, respectively, and a second site, IHRDLAARN (SEQID NO: 21), beginning at amino acids 652 and 642, respectively. Thesesites are characteristic of protein tyrosine kinases (Hanks, et al.,supra). The ECD of Rse contains 35% sequence identity with human Axl,which contains two immunoglobulin-like (IgL) repeats followed by twofibronectin type III (FNIII) repeats (FIG. 4). The conserved cysteineand tryptophan residues that are characteristic of IgL domains arepresent in human and murine Rse (FIG. 4). These features indicate thatAxl and Rse share a similar organization of structural domains in theECD, and that Rse represents the second member of the Axl family ofrPTKs. It is noted that Axl contains a unique sequence in the tyrosinekinase domain (KWIAIE (SEQ ID NO: 22)) that has been used to distinguishit from other kinases. [(K/T)W(T/M)APE (SEQ. ID NO: 23)]. In this sameposition, Rse contains the sequence [KWLALE (SEQ ID NO: 24)] which issimilar to Axl, but more similar to the HGFr (KWMALE (SEQ ID NO: 25)).

[0298] A distinguishing feature of the Axl/Rse family of rPTKs is theunique juxtaposition of IgL and FN-type III domains in the ECD. Axl andRse contain two membrane distal IgL repeats and two membrane proximalFN-type III repeats. The amino acid identity of human Axl and Rse in thefirst and second IgL repeats is 33% and 58% respectively, and 36% and42% in first and second FN-type III domains, respectively. A similarlevel of amino acid identity is observed in comparison of the murine Axland Rse IgL and FNIII domains. Without being limited to any one theory,it is believed that the combination of IgL and FNIII domains in the ECDof Rse may suggest that this protein plays a role in cellular adhesion.Cell adhesion molecules are grouped into either the immunoglobulinsuperfamily or the cadherin family based on homology and analysis ofbinding properties. The cadherins mediate cell-cell adhesion in acalcium dependent manner (Takeichi et al., Annu. Rev. Biochem., 59:237-252 [1990]). Cadherins associate with the actin cytoskeleton throughtheir intracellular domains via bridging proteins termed catenins (Ozawaet al., EMBO J., 8: [1989]). Cell adhesion mediated by members of theimmunoglobulin superfamily is calcium-independent.

[0299] Recently, the rPTK Dtrk (Pulido et al., EMBO J., 11:391-304[1992]), and the receptor protein phosphatase rPTPμ have been shown topromote cell adhesion in a calcium-independent homophilic manner(Brady-Kalnay et al., J. Cell. Biol., 122:961-972 [1993]). Brady-Kalnayet al. have suggested that a ligand for rPTPμ may be the ECD of the sametype of receptor on an adjacent cell. The interaction of the ECDs is notdependent on, nor appears to affect the properties of, the phosphataseactivity of the receptor. The ECDs LS of human and murine Rse containmultiple consensus sites for N-linked glycosylation (NxS/T), suggestingthat Rse is glycosylated (FIGS. 1A and 1B).

[0300] B. Construction of Cell Lines Expressing gD-Rse

[0301] To facilitate the analysis of the Rse protein, an epitope-taggedversion (referred to herein as gD-Rse) was constructed. The codingsequence for the 40 amino acid signal sequence of Rse was replaced witha sequence encoding amino acids 1-53 of the herpes simplex virus type I(HSV I) glycoprotein D (gD) [Lasky, L. A., et al., DNA 3: 23-29 (1984);and Paborsky, L. R. et al. Pro. Eng. 3: 547-553 (1990)]. Amino acids1-25 encode the signal sequence of gD while amino acids 26-56 contain anepitope for the monoclonal antibody 5B6. Oligos(5′-CAGCTGCTCGAGGCAGGTCTGAAGCTCATG (SEQ ID NO: 26), and5′-GCATGAATTCATGGCACACCTTCTACCGTG (SEQ ID NO: 27)) were used to add aXho I site to the human Rse cDNA by PCR. The gD-Rse cDNA was insertedinto the CMV-based expression vector pRK5 (Suva, L. J et al., Science,237: 893-896 [1987]). NIH3T3 cells were transfected with the gD-Rseexpression vector and the vector pCMV-Neo using a modified CaPO4protocol (Gorman, C., DNA Cloning: A Practical Approach, vol II:143-190, Glover, D. M., ed, IRL Press, Washington D.C. [1985]). After 9days, individual G418 resistant clones were picked and expanded.

[0302] To identify clones expressing gD-Rse, the anti-gD monoclonalantibody 5B6 was used to immunoprecipitate proteins from lysatesprepared from candidate clones. Immunoprecipitates were fractionated ona 7% SDS-polyacrylamide gel under reducing conditions, and Western blotsprepared from the gels were probed with the 5B6 antibody. A stableclone, 3T3.gD.R11, was isolated that expressed novel proteins of 120 kDaand 140 kDa that were reactive with 5B6 and not expressed in theparental 3T3 cells (FIG. 5, lanes 1 and 2). The predicted molecularweight of gD-Rse is approximately 96 kDa. The ECD of human Rse contains7 potential sites for N-linked glycosylation, and is glycosylated. Thus,while not being limited to any one theory, it is possible that the 120kDa and 140 kDa forms represent different glycoforms of gD-Rse.Alternatively, the 120 kDa form may represent a proteolyticallyprocessed form of gD-Rse. Fluorescence activated cell sorting using theanti-gD monoclonal antibody 5B6 confirmed the presence of the gD epitopeat the cell membrane.

[0303] C. Analysis of Tyrosine Kinase Activity of gD-Rse

[0304] The generally accepted mechanism by which ligands activate rPTKsinvolves ligand induced dimerization (Schlessinger, J., and Ullrich, A.,Neuron 9: 383-391 [1992]; Ullrich, A., and Schlessinger, J., Cell 61:203-212 [1990]; and Pazin, M. J., and Williams, L. T. TIBS 17: 374-378′[1992]). In some cases, rPTKs can be activated by antibodies directed tothe receptor ECD (Yarden, Y. Proc. Natl. Acad. Sci. U.S.A. 87: 2569-2573[1990]; McClain, D. A. J. Biol. Chem. 265: 21363-21367 [1990] and Sarup,J. C., Growth Regul. 1: 72-82 [1991]). It is believed that thesebivalent antibodies mimic ligand-induced activation by promotingreceptor oligomerization. It was determined if an antibody (i.e.,monoclonal antibody 5B6) to the epitope tag of gD-Rse could function asan agonist. Serum starved 3T3.gD.R11 or control NIH3T3 cells wereexposed to 5B6 monoclonal antibody, or a control antibody, for 10minutes. Using an anti-phosphotyrosine antibody (5E2) to probe Westernblots of immunoprecipitated lysates, an increase in phosphorylation ofthe 140 kDa form of gD-Rse in 3T3.gD.R11 cells treated with 5B6 wasclearly detected (FIG. 5, lanes 8 and 10). NIH3T3 cells and 3T3.gD.R11cells were plated at a density of 2×10⁶ cells per 60 mm dish inDMEM:F-12 (50:50)+10% FBS+glutamine+G418 media. After 16 hours, themedia was replaced with serum-free media for 2 hours, and thenantibodies were added at a concentration of 500 ng/ml. Cells wereharvested, lysates were immunoprecipitated with the 5B6 antibody,fractionated by SDS-PAGE, and Western blots were probed with theindicated antibodies as described (Lokker, N. A. et al., EMBO11:2503-2510 [1992]).

[0305] As discussed below, only minor differences in phosphorylation ofthe 120 kDa band were observed following treatment of 3T3.gD.R11 cellswith 5B6 antibody. The amount of phosphorylation of the 140 kDa band wasnot affected by treatment of 3T3.gD.R11 cells with control antibody(FIG. 5, lane 12). As an additional control, the blots were stripped andreprobed with the 5B6 monoclonal antibody to show that the amount ofgD-Rse loaded on the gel was similar (FIG. 5, lanes 2, 4, and 6). Asexpected, the increased phosphorylation of the 140 kDa gD-Rse proteinwas not observed in control NIH3T3 cells treated with either the 5B6 orcontrol antibody (FIG. 5, lanes 7, 9, and 11). Thus, it was concludedthat the tyrosine kinase domain of Rse is functional and that it can beregulated by receptor oligomerization. A time course experiment showedthat the kinetics of antibody-induced autophosphorylation were similarto those observed with other rPTKs; induction was observed within 10minutes, and declined gradually over the next 1-2 hours (FIG. 6). Theseresults indicate that dimerization of the Rse receptor is sufficient toinduce intrinsic tyrosine kinase activity. Considerably lessantibody-induced autophosphorylation of the 120 kDa form of gD-Rse thanof the 140 kDa form was detected (FIGS. 5 and 6). Both forms areexpressed at similar levels in the 3T3.gD.R11 cells, and both containthe gD epitope. There are a number of potential explanations for thisobservation. For example, without being limited to any one theory, the120 kDa gD-Rse might not be localized to the cell membrane. FACSanalysis suggests that at least a portion of the gD-epitope is localizedat the cell surface. However, these studies do not distinguish therelative ratios of the 120 kDa and 140 kDa forms at the membrane.

[0306] D. Northern Analysis

[0307] The expression of Rse was characterized using Northern blothybridization of polyadenylated RNA isolated from human tissues. Afragment from the portion of the cDNA-encoding the ECD was used as aprobe to minimize the possibility of cross-reaction with other tyrosinekinases. The human Rse probe was a 485 bp Pst I fragment correspondingto nucleotides 195-680 (FIG. 1A). Northern blots containing 2 μg ofpolyadenylated RNA from various human tissues or cell lines werehybridized with random-primed probes, washed and exposed according toconditions as described by the manufacturer Clontech, Palo Alto. The RNAblot shown in FIG. 7 was purchased from Clontech, Palo Alto, Calif. As acontrol for integrity of the RNA, the blots were stripped and reprobedwith a 2 Kb human b-actin DNA fragment (Clontech, Palo Alto Calif.). Theprobe detected a single predominant band of approximately 4.0 Kb (FIG.7A). The highest amount of hybridization was detected in samples of RNAfrom the brain and kidney, with lower expression observed in breast,heart, placenta, liver, lung, skeletal muscle, and pancreas. Probing thesame blot with a control human b-actin cDNA confirmed the integrity ofthe RNA in all of the samples (FIG. 7B). In other human tissues thatwere examined, Rse was expressed at high levels in the breast and at lowlevels in the adrenal gland and the large and small intestine. See Table2 below. TABLE 2 Expression of Rse mRNA in Human Tissues and Cell LinesExpression Level^(a) Tissue Breast +++ Adrenal + Large Intestine + SmallIntestine + Cell Line CMK11-5 ++ DAMI ++ THP-1 − Hep 3B +++ RAJI −K562 + MCF 7 + U937 +

[0308] The expression of Rse in various human cell lines was alsoanalyzed. Little, or no, Rse mRNA was detected by Northern blotting ofmRNA samples from the monocyte cell line THP-1 or the lymphoblast-likeRAJI cells (Table 2). However, the Rse transcript was detected in anumber of hematopoietic cell lines, including cells of the myeloid(i.e., myelogenous leukemia line K562 and myelomonocytic U937 cells) andthe megakaryocytic leukemia lines DAMI and CMK11-5, and the human breastcarcinoma cell line MCF-7. In the cell lines examined, the highest levelof expression was observed in Hep 3B cells, a human hepatocarcinoma cellline.

[0309] E. Chromosomal Localization of Human Rse Gene

[0310] Primers corresponding to unique regions in the 3′ end of the Rsegene were used to amplify human DNAs present in a panel of human-CHOhybrid cell lines (FIG. 8). Chromosomal localization was performed usingtwo sets of primer pairs (Btk 3-1²⁷²⁴: 5′-CACTGAGCTGGCTGACTAAG (SEQ IDNO: 28), Btk 3-4: 5′-CCTGATAGGCTGGGTACTCC (SEQ ID NO: 29); Btk 3-2²⁸¹⁵:5′-AAGCCCGGACTGACCAAA (SEQ ID NO: 30), Btk 3-3: 5′-GTGCGGAATCAGAAAGATGG(SEQ ID NO: 31)) derived from unique sequence in the 3′-untranslatedregion of RSE, amplifying DNA from a panel of 25 human-hamster hybridcell lines containing full complement of the human genome (BIOS, NewHaven, Conn.). PCR was performed with 250 ng DNA and 50 pmol each of the5′ and 3′ primers, 50 mM KCl, 1.5 mM MgCl₂, 20 μg/ml gelatin, 0.2 mMdNTPs and 2.5 units Taq polymerase in a final volume of 100 μl. Cyclesof 94° C. for 30 sec, 60° C. for 30 sec and 72° C. for 30 sec wererepeated 30 times. A portion of each sample (15 μl) was electrophoresedthrough a 1.5% agarose gel and either visualized by ethidium bromidestaining or transferred to a nylon membrane and hybridized to a³²P-labeled Rse insert probe prior to 5 hour autoradiography. Positiveswere scored and compared to a matrix summary of human chromosomalmaterial present in each of the somatic cell hybrid, human control orhamster control DNAs. This analysis localized the Rse gene to humanchromosome 15.

[0311] F. Construction of Human Rse-IQG Fusion Protein

[0312] The coding sequence of the ECD of Rse was fused to that of thehuman IgG-γ1 heavy chain in a multi-step process. PCR was used togenerate a fragment with a unique BstEII site 3′ to the coding sequencesof the Rse amino acid 428. The 5′ primer (5′-TCAAGACAATGGAACCCA (SEQ IDNO: 32)) and the 3′ primer (5′-CATGGAATTCGGTGACCGATGTGCGGCTGTGAGGAG (SEQID NO: 33)) were used in a 100 μl reaction containing 20 mM Tris-HCl, pH8.2, 10 mM KCl, 6 mM (NH₄)₂SO₄, 1.5 mM MgCl₂, 0.1% Triton X-100, 200dNTPs and 1 U of Pfu DNA polymerase (Stratagene) and 50 pmol each of theforward primer and the reverse primer and 40 ng of pBS.bptk3.9, whichcontains a Rse cDNA insert containing most of the extracellular domain,as template. After thirty cycles of denaturation (95° C., 1 min),annealing (55° C., 30 secs) and extension (72° C., 1 min), the PCRproduct was purified using Geneclean (Bio101), digested with BamHI andBstEII and recovered from low-melting temperature agarose gels. The PCRproduct was joined to the human IgG-γ₁ heavy chain cDNA through a uniqueBstEII site in that construct (Mark et al., J. Cell. Biol., 267:26166-26171 [1992]). The resulting construct (termedpRK.bpTK3.1gG.Fusion) contained the coding sequences for amino acids375-428 of Rse joined to those encoding human IgG-γ₁ heavy chain. Theremaining portion of the Rse ECD (amino acids 1-374) was then added bylinkage through the Bam HI site in pRK.bpTK3.IgG.Fusion to yieldpRK.Rse.IgG. Sequencing of the construct was carried out as describedabove.

[0313] G. Establishment of Stable Cell Populations Expressing Rse-IgG

[0314] For stable populations, the cDNA encoding Rse-IgG was subclonedinto the episomal CMV-driven expression plasmid pCIS.EBON, a pRK5derivative disclosed in Cachianes et al., Bio. Techniques, 15: 225-259(1993), the disclosure of which is expressly incorporated herein byreference. Human fetal kidney 293 cells (obtained from ATCC, 12301Parklawn Drive, Rockville, Md., USA) were transfected by the calciumphosphate technique. Cell monolayers were incubated for four hours inthe presence of the DNA precipitate, glycerol shocked, and cultured inF12:DMEM (1:1) containing 2 mM glutamine, 10% fetal bovine serum,penicillin and streptomycin. After 48 hours, populations were replatedin media containing G418 to select for a stable population of cells.Conditioned media was collected from cells expressing Rse-IgG that hadbeen cultured in serum-free media for 72 hours in the absence of G418.

[0315] H. Analysis of Rse-IgG by Western Blotting

[0316] For the Western blot analysis, 72-hour conditioned media fromtransfected 293 cells was subjected to electrophoresis under reducingconditions on a 7% SDS-acrylamide gel. The gel was blotted ontonitrocellulose with a Pharmacia LKB Novablot Western transfer apparatus.The filter was blocked in 1× NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris-OH,pH 7.5, 0.05% Triton-X 100) with 0.25% gelatin overnight at roomtemperature and then incubated with an HRP-conjugated antibody to thehuman IgG Fc (ICN). The Western blot was developed by a chemiluminescentdetection system as described by the manufacturer (Amersham).

[0317] I. Purification and Analysis of Rse-IgG

[0318] Rse-IgG was purified by affinity chromatography on a protein Acolumn using procedures as described by Chamow, S. M., et al.,Biochemistry, 29:9885-9891 (1990) with the following minormodifications. Conditioned media collected from cells expressing theRse-IgG was adjusted to 0.1 M citrate pH 6.0 and loaded directly onto aprotein A column (Repligen). The column was washed with 0.1 M citrate,pH 6.0, and was eluted with 3 M MgCl₂ with 10% glycerol. Fractions werepooled and desalted on a PD-10 column, dialyzed and concentrated againstPBS. Protein concentrations were determined by an ELISA against humanIgG (Fc). The protein was analyzed for purity by Coomassie staining ofPAGE gels.

[0319] J. Generation of Rabbit Polyclonal Antisera Against Rse-IgG

[0320] Polyclonal antibodies were generated in New Zealand White rabbitsagainst Rse-IgG. 4 μg in 100 μL PBS was emulsified with 100 μL Freund'sadjuvant (complete adjuvant for the primary injection and incompleteadjuvant for all boosts). For the primary immunization and the firstboost, the protein was injected directly into the popliteal lymph nodes(Sigel et al., Methods Enzymol., 93, 3-12 [1983]). For subsequentboosts, the protein was injected into subcutaneous and intramuscularsites. 1.3 μg protein/kg body weight was injected every 3 weeks withbleeds taken 1 and 2 weeks following each boost.

[0321] K. Stimulation of 3T3.gD.R11 Cells with Anti-Rse-IgG PolyclonalAntisera

[0322] Serum starved 3T3.gD.R11 cells or NIH3T3 cells were exposed topre-immune serum or polyclonal antisera directed against Rse-IgG at a{fraction (1/200)} dilution for 10 minutes. The gD-Rse protein wasimmunoprecipitated from extracts using the anti-gD monoclonal antibody5B6, as described above in section B. Proteins were fractionated on a0.7% SDS-PAGE under reducing conditions and transferred tonitrocellulose. Phosphoproteins were detected with theanti-phosphotyrosine antibody 5E2, as described in section C above. Theresults are depicted in FIG. 9. As can be seen in the figure, treatmentof the 3T3.gD.R11 cells with anti-Rse ECD antisera stimulated thephosphorylation of the 140 kDa gD-Rse protein (lane 4). This increasewas not observed in cells treated with pre-immune sera.

[0323] L. Deposit of Materials

[0324] The following plasmid DNA has been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC): Plasmid DNA ATCC Accession No. Deposit Date Strain Designation“RSE” Nov. 4, 1993

[0325] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable deposit for 30 years fromthe date of deposit. The plasmid DNA will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the plasmid DNA to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the plasmid DNA to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 8860G 638).

[0326] The assignee of the present application has agreed that if thedeposited DNA should be lost or destroyed when transformed into asuitable host cultivated under suitable conditions, it will be promptlyreplaced on notification with a specimen of the same DNA. Availabilityof the deposited DNA is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

EXAMPLE 2 Isolation and Characterization of HPTK6

[0327] A. cDNA Cloning and Sequencing

[0328] Degenerate oligodeoxyribonucleotide primers designed to sequencesencoding conserved amino acids in tyrosine kinases were used to isolatea primer which was used to screen a liver carcinoma (Hep 3B) cDNA lambdalibrary (see the procedures set forth in Example 1). Two full lengthclones encoding HPTK6 were found which differed in their 3′ untranslatedDNA sequences.

[0329] The assembled nucleotide and deduced amino acid sequences ofhuman HPTK6 are shown in FIG. 2. The HPTK6 cDNA sequence contains anopen reading frame of 913 amino acids. The mature form of human HPTK6 ispredicted to contain an ECD of 417 amino acids (i.e., amino acidresidues 19 to 417, shown in FIG. 2) and an ICD of 473 amino acids(i.e., amino acid residues 440 to 913 shown in FIG. 2). The sequenceappears to be substantially homologous to the human rPTK called DDRdisclosed by Johnson et al., supra, sharing 99.5% overall sequenceidentity therewith.

[0330] Similarly, the sequence shared 93.1% overall sequence identitywith the rPTK termed NEP, which appears to be the murine equivalent ofHPTK6 (see Zerlin et al., supra).

[0331] B. Northern Analysis

[0332] The expression of HPTK6 was characterized via Northern blothybridization of polyadenylated RNA isolated from human tissues. A 611base pair fragment from the portion of the cDNA encoding the ECD wasused as a probe to minimize the possibility of cross-reaction with othertyrosine kinases. Northern blots containing 2 μg of polyadenylated RNAfrom various human tissues or cell lines were hybridized withrandom-primed probes, washed and exposed as described by themanufacturer Clontech, Palo Alto, Calif. The RNA blots shown for humanadult and human fetal tissues in FIG. 10 were purchased from Clontech,Palo Alto, Calif. The probe detected a single predominant band of3.8-3.9 Kb (FIG. 10). In the human adult tissues, the highest amount ofhybridization was detected in samples of RNA from the kidney andplacenta, with lower expression observed in the brain, lung, skeletalmuscle and pancreas. No expression in the liver was detected. See FIG.10A. Expression of HPTK6 in the fetal tissues was different fromexpression in the adult tissues. With reference to FIG. 10B, the highestexpression was observed in the fetal brain with lower expressionevidenced in the fetal kidney and lung tissue, respectively. Like theadult tissue, no expression in the liver was observed. In the adult andfetal tissues studied, the expression of HPTK6 was generally low.

[0333] Expression of HPTK6 in murine tissue was also investigated. TheRNA blot shown in FIGS. 11A and B was obtained from Clontech, Palo Alto,Calif. As a control for integrity of the RNA, the blots were strippedand reprobed with a 2 Kb human b-actin DNA fragment (Clontech, Palo AltoCalif.), FIG. 11B. Bands of about 4.0 and 4.3 Kb were detected. Thehighest amount of hybridization was detected in samples of RNA from thekidney and brain, with lower expression observed in the testis, spleenand lung.

[0334] Expression of HPTK6 in various cell lines was also studied viaNorthern blotting of mRNA samples from these cell lines. The results ofthese experiments are shown in Table 3 below. TABLE 3 Expression ofHPTK6 mRNA in Human Cell Lines Cell Line Expression Level^(a) MCF 7(human breast ++ carcinoma) Thymoma ++ Hep 3B (liver + carcinoma)CMK11-5 (megakaryocyte − progenitor) DAMI (megakaryocyte − progenitor)BMMC (bone marrow − mononucleocytes) PBMC (peripheral blood −mononuclear cells) Megakaryoblast −

[0335] In situ hybridization of HPTK6 RNA in deparaffinized sections ofhuman and murine embryos was performed according to Haub & Goldfarb,Development, 112: 396-406 [1991], using ³⁵S-labeled CRNA riboprobes. DNAfragments from HPTK6 cDNA served as templates for synthesis of sense(+ve) and antisense (−) riboprobes. Hybridized slides were subjected toautoradiography. The traverse sections of human and mouse fetal tissuesare shown in FIGS. 12A-C. Antisense riboprobes gave signals, while senseprobes gave no signal. As indicated in FIG. 12, high levels ofexpression were observed in the fetal brain and spinal cord for both themouse and human.

[0336] These results indicate that HPTK6 may play a role in cancerformation in certain cells, e.g., human breast carcinoma cells.Accordingly, antagonist ligands to the receptor may be useful for cancertherapies. The high level of expression in fetal brain indicates thatHPTK6, or its ligands, may be useful for treating neurodegenerativediseases as discussed earlier herein.

1 35 3611 bases nucleic acid single linear 1 CCGCCGATGG CGCTGAGGCGGAGCATGGGG CGGCCGGGGC TCCCGCCGCT 50 GCCGCTGCCG CCGCCACCGC GGCTCGGGCTGCTGCTGGCG GCTCTGGCTT 100 CTCTGCTGCT CCCGGAGTCC GCCGCCGCAG GTCTGAAGCTCATGGGAGCC 150 CCGGTGAAGC TGACAGTGTC TCAGGGGCAG CCGGTGAAGC TCAACTGCAG200 TGTGGAGGGG ATGGAGGAGC CTGACATCCA GTGGGTGAAG GATGGGGCTG 250TGGTCCAGAA CTTGGACCAG TTGTACATCC CAGTCAGCGA GCAGCACTGG 300 ATCGGCTTCCTCAGCCTGAA GTCAGTGGAG CGCTCTGACG CCGGCCGGTA 350 CTGGTGCCAG GTGGAGGATGGGGGTGAAAC CGAGATCTCC CAGCCAGTGT 400 GGCTCACGGT AGAAGGTGTG CCATTTTTCACAGTGGAGCC AAAAGATCTG 450 GCAGTGCCAC CCAATGCCCC TTTCCAACTG TCTTGTGAGGCTGTGGGTCC 500 CCCTGAACCT GTTACCATTG TCTGGTGGAG AGGAACTACG AAGATCGGGG550 GACCCGCTCC CTCTCCATCT GTTTTAAATG TAACAGGGGT GACCCAGAGC 600ACCATGTTTT CCTGTGAAGC TCACAACCTA AAAGGCCTGG CCTCTTCTCG 650 CACAGCCACTGTTCACCTTC AAGCACTGCC TGCAGCCCCC TTCAACATCA 700 CCGTGACAAA GCTTTCCAGCAGCAACGCTA GTGTGGCCTG GATGCCAGGT 750 GCTGATGGCC GAGCTCTGCT ACAGTCCTGTACAGTTCAGG TGACACAGGC 800 CCCAGGAGGC TGGGAAGTCC TGGCTGTTGT GGTCCCTGTGCCCCCCTTTA 850 CCTGCCTGCT CCGGGACCTG GTGCCTGCCA CCAACTACAG CCTCAGGGTG900 CGCTGTGCCA ATGCCTTGGG GCCCTCTCCC TATGCTGACT GGGTGCCCTT 950TCAGACCAAG GGTCTAGCCC CAGCCAGCGC TCCCCAAAAC CTCCATGCCA 1000 TCCGCACAGATTCAGGCCTC ATCTTGGAGT GGGAAGAAGT GATCCCCGAG 1050 GCCCCTTTGG AAGGCCCCCTGGGACCCTAC AAACTGTCCT GGGTTCAAGA 1100 CAATGGAACC CAGGATGAGC TGACAGTGGAGGGGACCAGG GCCAATTTGA 1150 CAGGCTGGGA TCCCCAAAAG GACCTGATCG TACGTGTGTGCGTCTCCAAT 1200 GCAGTTGGCT GTGGACCCTG GAGTCAGCCA CTGGTGGTCT CTTCTCATGA1250 CCGTGCAGGC CAGCAGGGCC CTCCTCACAG CCGCACATCC TGGGTACCTG 1300TGGTCCTTGG TGTGCTAACG GCCCTGGTGA CGGCTGCTGC CCTGGCCCTC 1350 ATCCTGCTTCGAAAGAGACG GAAAGAGACG CGGTTTGGGC AAGCCTTTGA 1400 CAGTGTCATG GCCCGGGGAGAGCCAGCCGT TCACTTCCGG GCAGCCCGGT 1450 CCTTCAATCG AGAAAGGCCC GAGCGCATCGAGGCCACATT GGACAGCTTG 1500 GGCATCAGCG ATGAACTAAA GGAAAAACTG GAGGATGTGCTCATCCCAGA 1550 GCAGCAGTTC ACCCTGGGCC GGATGTTGGG CAAAGGAGAG TTTGGTTCAG1600 TGCGGGAGGC CCAGCTGAAG CAAGAGGATG GCTCCTTTGT GAAAGTGGCT 1650GTGAAGATGC TGAAAGCTGA CATCATTGCC TCAAGCGACA TTGAAGAGTT 1700 CCTCAGGGAAGCAGCTTGCA TGAAGGAGTT TGACCATCCA CACGTGGCCA 1750 AACTTGTTGG GGTAAGCCTCCGGAGCAGGG CTAAAGGCCG TCTCCCCATC 1800 CCCATGGTCA TCTTGCCCTT CATGAAGCATGGGGACCTGC ATGCCTTCCT 1850 GCTCGCCTCC CGGATTGGGG AGAACCCCTT TAACCTACCCCTCCAGACCC 1900 TGATCCGGTT CATGGTGGAC ATTGCCTGCG GCATGGAGTA CCTGAGCTCT1950 CGGAACTTCA TCCACCGAGA CCTGGCTGCT CGGAATTGCA TGCTGGCAGA 2000GGACATGACA GTGTGTGTGG CTGACTTCGG ACTCTCCCGG AAGATCTACA 2050 GTGGGGACTACTATCGTCAA GGCTGTGCCT CCAAACTGCC TGTCAAGTGG 2100 CTGGCCCTGG AGAGCCTGGCCGACAACCTG TATACTGTGC AGAGTGACGT 2150 GTGGGCGTTC GGGGTGACCA TGTGGGAGATCATGACACGT GGGCAGACGC 2200 CATATGCTGG CATCGAAAAC GCTGAGATTT ACAACTACCTCATTGGCGGG 2250 AACCGCCTGA AACAGCCTCC GGAGTGTATG GAGGACGTGT ATGATCTCAT2300 GTACCAGTGC TGGAGTGCTG ACCCCAAGCA GCGCCCGAGC TTTACTTGTC 2350TGCGAATGGA ACTGGAGAAC ATCTTGGGCC AGCTGTCTGT GCTATCTGCC 2400 AGCCAGGACCCCTTATACAT CAACATCGAG AGAGCTGAGG AGCCCACTGC 2450 GGGAGGCAGC CTGGAGCTACCTGGCAGGGA TCAGCCCTAC AGTGGGGCTG 2500 GGGATGGCAG TGGCATGGGG GCAGTGGGTGGCACTCCCAG TGACTGTCGG 2550 TACATACTCA CCCCCGGAGG GCTGGCTGAG CAGCCAGGGCAGGCAGAGCA 2600 CCAGCCAGAG AGTCCCCTCA ATGAGACACA GAGGCTTTTG CTGCTGCAGC2650 AAGGGCTACT GCCACACAGT AGCTGTTAGC CCACAGGCAG AGGGCATCGG 2700GGCCATTTGG CCGGCTCTGG TGGCCACTGA GCTGGCTGAC TAAGCCCCGT 2750 CTGACCCCAGCCCAGACAGC AAGGTGTGGA GGCTCCTGTG GTAGTCCTCC 2800 CAAGCTGTGC TGGGAAGCCCGGACTGACCA AATCACCCAA TCCCAGTTCT 2850 TCCTGCAACC ACTCTGTGGC CAGCCTGGCATCAGTTTAGG CCTTGGCTTG 2900 ATGGAAGTGG GCCAGTCCTG GTTGTCTGAA CCCAGGCAGCTGGCAGGAGT 2950 GGGGTGGTTA TGTTTCCATG GTTACCATGG GTGTGGATGG CAGTGTGGGG3000 AGGGCAGGTC CAGCTCTGTG GGCCCTACCC TCCTGCTGAG CTGCCCCTGC 3050TGCTTAAGTG CATGCATTGA GCTGCCTCCA GCCTGGTGGC CCAGCTATTA 3100 CCACACTTGGGGTTTAAATA TCCAGGTGTG CCCCTCCAAG TCACAAAGAG 3150 ATGTCCTTGT AATATTCCCTTTTAGGTGAG GGTTGGTAAG GGGTTGGTAT 3200 CTCAGGTCTG AATCTTCACC ATCTTTCTGATTCCGCACCC TGCCTACGCC 3250 AGGAGAAGTT GAGGGGAGCA TGCTTCCCTG CAGCTGACCGGGTCACACAA 3300 AGGCATGCTG GAGTACCCAG CCTATCAGGT GCCCCTCTTC CAAAGGCAGC3350 GTGCCGAGCC AGCAAGAGGA AGGGGTGCTG TGAGGCTTGC CCAGGAGCAA 3400GTGAGGCCGG AGAGGAGTTC AGGAACCCTT CTCCATACCC ACAATCTGAG 3450 CACGCTACCAAATCTCAAAA TATCCTAAGA CTAACAAAGG CAGCTGTGTC 3500 TGAGCCCAAC CCTTCTAAACGGTGACCTTT AGTGCCAACT TCCCCTCTAA 3550 CTGGACAGCC TCTTCTGTCC CAAGTCTCCAGAGAGAAATC AGGCCTGATG 3600 AGGGGGAATT C 3611 890 amino acids amino acidlinear 2 Met Ala Leu Arg Arg Ser Met Gly Arg Pro Gly Leu Pro Pro Leu 1 510 15 Pro Leu Pro Pro Pro Pro Arg Leu Gly Leu Leu Leu Ala Ala Leu 20 2530 Ala Ser Leu Leu Leu Pro Glu Ser Ala Ala Ala Gly Leu Lys Leu 35 40 45Met Gly Ala Pro Val Lys Leu Thr Val Ser Gln Gly Gln Pro Val 50 55 60 LysLeu Asn Cys Ser Val Glu Gly Met Glu Glu Pro Asp Ile Gln 65 70 75 Trp ValLys Asp Gly Ala Val Val Gln Asn Leu Asp Gln Leu Tyr 80 85 90 Ile Pro ValSer Glu Gln His Trp Ile Gly Phe Leu Ser Leu Lys 95 100 105 Ser Val GluArg Ser Asp Ala Gly Arg Tyr Trp Cys Gln Val Glu 110 115 120 Asp Gly GlyGlu Thr Glu Ile Ser Gln Pro Val Trp Leu Thr Val 125 130 135 Glu Gly ValPro Phe Phe Thr Val Glu Pro Lys Asp Leu Ala Val 140 145 150 Pro Pro AsnAla Pro Phe Gln Leu Ser Cys Glu Ala Val Gly Pro 155 160 165 Pro Glu ProVal Thr Ile Val Trp Trp Arg Gly Thr Thr Lys Ile 170 175 180 Gly Gly ProAla Pro Ser Pro Ser Val Leu Asn Val Thr Gly Val 185 190 195 Thr Gln SerThr Met Phe Ser Cys Glu Ala His Asn Leu Lys Gly 200 205 210 Leu Ala SerSer Arg Thr Ala Thr Val His Leu Gln Ala Leu Pro 215 220 225 Ala Ala ProPhe Asn Ile Thr Val Thr Lys Leu Ser Ser Ser Asn 230 235 240 Ala Ser ValAla Trp Met Pro Gly Ala Asp Gly Arg Ala Leu Leu 245 250 255 Gln Ser CysThr Val Gln Val Thr Gln Ala Pro Gly Gly Trp Glu 260 265 270 Val Leu AlaVal Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu 275 280 285 Arg Asp LeuVal Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys 290 295 300 Ala Asn AlaLeu Gly Pro Ser Pro Tyr Ala Asp Trp Val Pro Phe 305 310 315 Gln Thr LysGly Leu Ala Pro Ala Ser Ala Pro Gln Asn Leu His 320 325 330 Ala Ile ArgThr Asp Ser Gly Leu Ile Leu Glu Trp Glu Glu Val 335 340 345 Ile Pro GluAla Pro Leu Glu Gly Pro Leu Gly Pro Tyr Lys Leu 350 355 360 Ser Trp ValGln Asp Asn Gly Thr Gln Asp Glu Leu Thr Val Glu 365 370 375 Gly Thr ArgAla Asn Leu Thr Gly Trp Asp Pro Gln Lys Asp Leu 380 385 390 Ile Val ArgVal Cys Val Ser Asn Ala Val Gly Cys Gly Pro Trp 395 400 405 Ser Gln ProLeu Val Val Ser Ser His Asp Arg Ala Gly Gln Gln 410 415 420 Gly Pro ProHis Ser Arg Thr Ser Trp Val Pro Val Val Leu Gly 425 430 435 Val Leu ThrAla Leu Val Thr Ala Ala Ala Leu Ala Leu Ile Leu 440 445 450 Leu Arg LysArg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe Asp 455 460 465 Ser Val MetAla Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala 470 475 480 Arg Ser PheAsn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu 485 490 495 Asp Ser LeuGly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp 500 505 510 Val Leu IlePro Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly 515 520 525 Lys Gly GluPhe Gly Ser Val Arg Glu Ala Gln Leu Lys Gln Glu 530 535 540 Asp Gly SerPhe Val Lys Val Ala Val Lys Met Leu Lys Ala Asp 545 550 555 Ile Ile AlaSer Ser Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala 560 565 570 Cys Met LysGlu Phe Asp His Pro His Val Ala Lys Leu Val Gly 575 580 585 Val Ser LeuArg Ser Arg Ala Lys Gly Arg Leu Pro Ile Pro Met 590 595 600 Val Ile LeuPro Phe Met Lys His Gly Asp Leu His Ala Phe Leu 605 610 615 Leu Ala SerArg Ile Gly Glu Asn Pro Phe Asn Leu Pro Leu Gln 620 625 630 Thr Leu IleArg Phe Met Val Asp Ile Ala Cys Gly Met Glu Tyr 635 640 645 Leu Ser SerArg Asn Phe Ile His Arg Asp Leu Ala Ala Arg Asn 650 655 660 Cys Met LeuAla Glu Asp Met Thr Val Cys Val Ala Asp Phe Gly 665 670 675 Leu Ser ArgLys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Cys 680 685 690 Ala Ser LysLeu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala 695 700 705 Asp Asn LeuTyr Thr Val Gln Ser Asp Val Trp Ala Phe Gly Val 710 715 720 Thr Met TrpGlu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly 725 730 735 Ile Glu AsnAla Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg 740 745 750 Leu Lys GlnPro Pro Glu Cys Met Glu Asp Val Tyr Asp Leu Met 755 760 765 Tyr Gln CysTrp Ser Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr 770 775 780 Cys Leu ArgMet Glu Leu Glu Asn Ile Leu Gly Gln Leu Ser Val 785 790 795 Leu Ser AlaSer Gln Asp Pro Leu Tyr Ile Asn Ile Glu Arg Ala 800 805 810 Glu Glu ProThr Ala Gly Gly Ser Leu Glu Leu Pro Gly Arg Asp 815 820 825 Gln Pro TyrSer Gly Ala Gly Asp Gly Ser Gly Met Gly Ala Val 830 835 840 Gly Gly ThrPro Ser Asp Cys Arg Tyr Ile Leu Thr Pro Gly Gly 845 850 855 Leu Ala GluGln Pro Gly Gln Ala Glu His Gln Pro Glu Ser Pro 860 865 870 Leu Asn GluThr Gln Arg Leu Leu Leu Leu Gln Gln Gly Leu Leu 875 880 885 Pro His SerSer Cys 890 3637 bases nucleic acid single linear 3 GAATTCTCGAGTCGACGTTG GACTTGAAGG AATGCCAAGA GATGCTGCCC 50 CCACCCCCTT AGGCCCGAGGGATCAGGAGC TATGGGACCA GAGGCCCTGT 100 CATCTTTACT GCTGCTGCTC TTGGTGGCAAGTGGAGATGC TGACATGAAG 150 GGACATTTTG ATCCTGCCAA GTGCCGCTAT GCCCTGGGCATGCAGGACCG 200 GACCATCCCA GACAGTGACA TCTCTGCTTC CAGCTCCTGG TCAGATTCCA250 CTGCCGCCCG CCACAGCAGG TTGGAGAGCA GTGACGGGGA TGGGGCCTGG 300TGCCCCGCAG GGTCGGTGTT TCCCAAGGAG GAGGAGTACT TGCAGGTGGA 350 TCTACAACGACTGCACCTGG TGGCTCTGGT GGGCACCCAG GGACGGCATG 400 CCGGGGGCCT GGGCAAGGAGTTCTCCCGGA GCTACCGGCT GCGTTACTCC 450 CGGGATGGTC GCCGCTGGAT GGGCTGGAAGGACCGCTGGG GTCAGGAGGT 500 GATCTCAGGC AATGAGGACC CTGAGGGAGT GGTGCTGAAGGACCTTGGGC 550 CCCCCATGGT TGCCCGACTG GTTCGCTTCT ACCCCCGGGC TGACCGGGTC600 ATGAGCGTCT GTCTGCGGGT AGAGCTCTAT GGCTGCCTCT GGAGGGATGG 650ACTCCTGTCT TACACCGCCC CTGTGGGGCA GACAATGTAT TTATCTGAGG 700 CCGTGTACCTCAACGACTCC ACCTATGACG GACATACCGT GGGCGGACTG 750 CAGTATGGGG GTCTGGGCCAGCTGGCAGAT GGTGTGGTGG GGCTGGATGA 800 CTTTAGGAAG AGTCAGGAGC TGCGGGTCTGGCCAGGCTAT GACTATGTGG 850 GATGGAGCAA CCACAGCTTC TCCAGTGGCT ATGTGGAGATGGAGTTTGAG 900 TTTGACCGGC TGAGGGCCTT CCAGGCTATG CAGGTCCACT GTAACAACAT950 GCACACGCTG GGAGCCCGTC TGCCTGGCGG GGTGGAATGT CGCTTCCGGC 1000GTGGCCCTGC CATGGCCTGG GAGGGGGAGC CCATGCGCCA CAACCTAGGG 1050 GGCAACCTGGGGGACCCCAG AGCCCGGGCT GTCTCAGTGC CCCTTGGCGG 1100 CCGTGTGGCT CGCTTTCTGCAGTGCCGCTT CCTCTTTGCG GGGCCCTGGT 1150 TACTCTTCAG CGAAATCTCC TTCATCTCTGATGTGGTGAA CAATTCCTCT 1200 CCGGCACTGG GAGGCACCTT CCCGCCAGCC CCCTGGTGGCCGCCTGGCCC 1250 ACCTCCCACC AACTTCAGCA GCTTGGAGCT GGAGCCCAGA GGCCAGCAGC1300 CCGTGGCCAA GCCCGAGGGG AGCCCGACCG CCATCCTCAT CGGCTGCCTG 1350GTGGCCATCA TCCTGCTCCT GCTGCTCATC ATTGCCCTCA TGCTCTGGCG 1400 GCTGCACTGGCGCAGGCTCC TCAGCAAGGC TGAACGGAGG GTGTTGGAAG 1450 AGGAGCTGAC GGTTCACCTCTCTGTCCCTG GGGACACTAT CCTCATCAAC 1500 AACCGCCCAG GTCCTAGAGA GCCACCCCCGTACCAGGAGC CCCGGCCTCG 1550 TGGGAATCCG CCCCACTCCG CTCCCTGTGT CCCCAATGGCTCTGCGTTGC 1600 TGCTCTCCAA TCCAGCCTAC CGCCTCCTTC TGGCCACTTA CGCCCGTCCC1650 CCTCGAGGCC CGGGCCCCCC CACACCCGCC TGGGCCAAAC CCACCAACAC 1700CCAGGCCTAC AGTGGGGACT ATATGGAGCC TGAGAAGCCA GGCGCCCCGC 1750 TTCTGCCCCCACCTCCCCAG AACAGCGTCC CCCATTATGC CGAGGCTGAC 1800 ATTGTTACCC TGCAGGGCGTCACCGGGGGC AACACCTATG CTGTGCCTGC 1850 ACTGCCCCCA GGGGCAGTCG GGGATGGGCCCCCCAGAGTG GATTTCCCTC 1900 GATCTCGACT CCGCTTCAAG GAGAAGCTTG GCGAGGGCCAGTTTGGGGAG 1950 GTGCACCTGT GTGAGGTCGA CAGCCCTCAA GATCTGGTCA GTCTTGATTT2000 CCCCCTTAAT GTGCGTAAGG GACACCCTTT GCTGGTAGCT GTCAAGATCT 2050TACGGCCAGA TGCCACCAAG AATGCCAGGA ATGATTTCCT GAAAGAGGTG 2100 AAGATCATGTCGAGGCTCAA GGACCCAAAC ATCATTCGGC TGCTGGGCGT 2150 GTGTGTGCAG GACGACCCCCTCTGCATGAT TACTGACTAC ATGGAGAACG 2200 GCGACCTCAA CCAGTTCCTC AGTGCCCACCAGCTGGAGGA CAAGGCAGCC 2250 GAGGGGGCCC CTGGGGACGG GCAGGCTGCG CAGGGGCCCACCATCAGCTA 2300 CCCAATGCTG CTGCATGTGG CAGCCCAGAT CGCCTCCGGC ATGCGCTATC2350 TGGCCACACT CAACTTTGTA CATCGGGACC TGGCCACGCG GAACTGCCTA 2400GTTGGGGAAA ATTTCACCAT CAAAATCGCA GACTTTGGCA TGAGCCGGAA 2450 CCTCTATGCTGGGGACTATT ACCGTGTGCA GGGCCGGGCA GTGCTGCCCA 2500 TCCGCTGGAT GGCCTGGGAGTGCATCCTCA TGGGGAAGTT CACGACTGCG 2550 AGTGACGTGT GGGCCTTTGG TGTGACCCTGTGGGAGGTGC TGATGCTCTG 2600 TAGGGCCCAG CCCTTTGGGC AGCTCACCGA CGAGCAGGTCATCGAGAACG 2650 CGGGGGAGTT CTTCCGGGAC CAGGGCCGGC AGGTGTACCT GTCCCGGCCG2700 CCTGCCTGCC CGCAGGGCCT ATATGAGCTG ATGCTTCGGT GCTGGAGCCG 2750GGAGTCTGAG CAGCGACCAC CCTTTTCCCA GCTGCATCGG TTCCTGGCAG 2800 AGGATGCACTCAACACGGTG TGAATCACAC ATCCAGCTGC CCCTCCCTCA 2850 GGGAGTGATC CAGGGGAAGCCAGTGACACT AAAACAAGAG GACACAATGG 2900 CACCTCTGCC CTTCCCCTCC CGACAGCCCATCACCTCTAA TAGAGGCAGT 2950 GAGACTGCAG AAGCCCCTGT CGCCCACCCA GCTGGTCCTGTGGATGGGAT 3000 CCTCTCCACC CTCCTCTAGC CATCCCTTGG GGAAGGGTGG GGAGAAATAT3050 AGGATAGACA CTGGACATGG CCCATTGGAG CACCTGGGCC CCACTGGACA 3100ACACTGATTC CTGGAGAGGT GGCTGCGCCC CCAGCTTCTC TCTCCCTGTC 3150 ACACACTGGACCCCACTGGC TGAGAATCTG GGGGTGAGGA GGACAAGAAG 3200 GAGAGGAAAA TGTTTCCTTGTGCCTGCTCC TGTACTTGTC CTCAGCTTGG 3250 GCTTCTTCCT CCTCCATCAC CTGAAACACTGGACCTGGGG GTAGCCCCGC 3300 CCCAGCCCTC AGTCACCCCC ACTTCCCACC TGCAGTCTTGTAGCTAGAAC 3350 TTCTCTAAGC CTATACGTTT CTGTGGAGTA AATATTGGGA TTGGGGGGAA3400 AGAGGGAGCA ACGGCCCATA GCCTTGGGGT TGGACATCTC TAGTGTAGCT 3450GCCACATTGA TTTTTCTATA ATCACTTGGG GTTTGTACAT TTTTGGGGGG 3500 AGAGACACAGATTTTTACAC TAATATATGG ACCTAGCTTG AGGCAATTTT 3550 AATCCCCTGC ACTAGGCAGGTAATAATAAA GGTTGAGTTT TCCACAAAAA 3600 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAA 3637 913 amino acids amino acid linear 4 Met Gly Pro Glu Ala LeuSer Ser Leu Leu Leu Leu Leu Leu Val 1 5 10 15 Ala Ser Gly Asp Ala AspMet Lys Gly His Phe Asp Pro Ala Lys 20 25 30 Cys Arg Tyr Ala Leu Gly MetGln Asp Arg Thr Ile Pro Asp Ser 35 40 45 Asp Ile Ser Ala Ser Ser Ser TrpSer Asp Ser Thr Ala Ala Arg 50 55 60 His Ser Arg Leu Glu Ser Ser Asp GlyAsp Gly Ala Trp Cys Pro 65 70 75 Ala Gly Ser Val Phe Pro Lys Glu Glu GluTyr Leu Gln Val Asp 80 85 90 Leu Gln Arg Leu His Leu Val Ala Leu Val GlyThr Gln Gly Arg 95 100 105 His Ala Gly Gly Leu Gly Lys Glu Phe Ser ArgSer Tyr Arg Leu 110 115 120 Arg Tyr Ser Arg Asp Gly Arg Arg Trp Met GlyTrp Lys Asp Arg 125 130 135 Trp Gly Gln Glu Val Ile Ser Gly Asn Glu AspPro Glu Gly Val 140 145 150 Val Leu Lys Asp Leu Gly Pro Pro Met Val AlaArg Leu Val Arg 155 160 165 Phe Tyr Pro Arg Ala Asp Arg Val Met Ser ValCys Leu Arg Val 170 175 180 Glu Leu Tyr Gly Cys Leu Trp Arg Asp Gly LeuLeu Ser Tyr Thr 185 190 195 Ala Pro Val Gly Gln Thr Met Tyr Leu Ser GluAla Val Tyr Leu 200 205 210 Asn Asp Ser Thr Tyr Asp Gly His Thr Val GlyGly Leu Gln Tyr 215 220 225 Gly Gly Leu Gly Gln Leu Ala Asp Gly Val ValGly Leu Asp Asp 230 235 240 Phe Arg Lys Ser Gln Glu Leu Arg Val Trp ProGly Tyr Asp Tyr 245 250 255 Val Gly Trp Ser Asn His Ser Phe Ser Ser GlyTyr Val Glu Met 260 265 270 Glu Phe Glu Phe Asp Arg Leu Arg Ala Phe GlnAla Met Gln Val 275 280 285 His Cys Asn Asn Met His Thr Leu Gly Ala ArgLeu Pro Gly Gly 290 295 300 Val Glu Cys Arg Phe Arg Arg Gly Pro Ala MetAla Trp Glu Gly 305 310 315 Glu Pro Met Arg His Asn Leu Gly Gly Asn LeuGly Asp Pro Arg 320 325 330 Ala Arg Ala Val Ser Val Pro Leu Gly Gly ArgVal Ala Arg Phe 335 340 345 Leu Gln Cys Arg Phe Leu Phe Ala Gly Pro TrpLeu Leu Phe Ser 350 355 360 Glu Ile Ser Phe Ile Ser Asp Val Val Asn AsnSer Ser Pro Ala 365 370 375 Leu Gly Gly Thr Phe Pro Pro Ala Pro Trp TrpPro Pro Gly Pro 380 385 390 Pro Pro Thr Asn Phe Ser Ser Leu Glu Leu GluPro Arg Gly Gln 395 400 405 Gln Pro Val Ala Lys Pro Glu Gly Ser Pro ThrAla Ile Leu Ile 410 415 420 Gly Cys Leu Val Ala Ile Ile Leu Leu Leu LeuLeu Ile Ile Ala 425 430 435 Leu Met Leu Trp Arg Leu His Trp Arg Arg LeuLeu Ser Lys Ala 440 445 450 Glu Arg Arg Val Leu Glu Glu Glu Leu Thr ValHis Leu Ser Val 455 460 465 Pro Gly Asp Thr Ile Leu Ile Asn Asn Arg ProGly Pro Arg Glu 470 475 480 Pro Pro Pro Tyr Gln Glu Pro Arg Pro Arg GlyAsn Pro Pro His 485 490 495 Ser Ala Pro Cys Val Pro Asn Gly Ser Ala LeuLeu Leu Ser Asn 500 505 510 Pro Ala Tyr Arg Leu Leu Leu Ala Thr Tyr AlaArg Pro Pro Arg 515 520 525 Gly Pro Gly Pro Pro Thr Pro Ala Trp Ala LysPro Thr Asn Thr 530 535 540 Gln Ala Tyr Ser Gly Asp Tyr Met Glu Pro GluLys Pro Gly Ala 545 550 555 Pro Leu Leu Pro Pro Pro Pro Gln Asn Ser ValPro His Tyr Ala 560 565 570 Glu Ala Asp Ile Val Thr Leu Gln Gly Val ThrGly Gly Asn Thr 575 580 585 Tyr Ala Val Pro Ala Leu Pro Pro Gly Ala ValGly Asp Gly Pro 590 595 600 Pro Arg Val Asp Phe Pro Arg Ser Arg Leu ArgPhe Lys Glu Lys 605 610 615 Leu Gly Glu Gly Gln Phe Gly Glu Val His LeuCys Glu Val Asp 620 625 630 Ser Pro Gln Asp Leu Val Ser Leu Asp Phe ProLeu Asn Val Arg 635 640 645 Lys Gly His Pro Leu Leu Val Ala Val Lys IleLeu Arg Pro Asp 650 655 660 Ala Thr Lys Asn Ala Arg Asn Asp Phe Leu LysGlu Val Lys Ile 665 670 675 Met Ser Arg Leu Lys Asp Pro Asn Ile Ile ArgLeu Leu Gly Val 680 685 690 Cys Val Gln Asp Asp Pro Leu Cys Met Ile ThrAsp Tyr Met Glu 695 700 705 Asn Gly Asp Leu Asn Gln Phe Leu Ser Ala HisGln Leu Glu Asp 710 715 720 Lys Ala Ala Glu Gly Ala Pro Gly Asp Gly GlnAla Ala Gln Gly 725 730 735 Pro Thr Ile Ser Tyr Pro Met Leu Leu His ValAla Ala Gln Ile 740 745 750 Ala Ser Gly Met Arg Tyr Leu Ala Thr Leu AsnPhe Val His Arg 755 760 765 Asp Leu Ala Thr Arg Asn Cys Leu Val Gly GluAsn Phe Thr Ile 770 775 780 Lys Ile Ala Asp Phe Gly Met Ser Arg Asn LeuTyr Ala Gly Asp 785 790 795 Tyr Tyr Arg Val Gln Gly Arg Ala Val Leu ProIle Arg Trp Met 800 805 810 Ala Trp Glu Cys Ile Leu Met Gly Lys Phe ThrThr Ala Ser Asp 815 820 825 Val Trp Ala Phe Gly Val Thr Leu Trp Glu ValLeu Met Leu Cys 830 835 840 Arg Ala Gln Pro Phe Gly Gln Leu Thr Asp GluGln Val Ile Glu 845 850 855 Asn Ala Gly Glu Phe Phe Arg Asp Gln Gly ArgGln Val Tyr Leu 860 865 870 Ser Arg Pro Pro Ala Cys Pro Gln Gly Leu TyrGlu Leu Met Leu 875 880 885 Arg Cys Trp Ser Arg Glu Ser Glu Gln Arg ProPro Phe Ser Gln 890 895 900 Leu His Arg Phe Leu Ala Glu Asp Ala Leu AsnThr Val 905 910 913 1164 bases nucleic acid single linear 5 GCAGGTCTGAAGCTCATGGG AGCCCCGGTG AAGCTGACAG TGTCTCAGGG 50 GCAGCCGGTG AAGCTCAACTGCAGTGTGGA GGGGATGGAG GAGCCTGACA 100 TCCAGTGGGT GAAGGATGGG GCTGTGGTCCAGAACTTGGA CCAGTTGTAC 150 ATCCCAGTCA GCGAGCAGCA CTGGATCGGC TTCCTCAGCCTGAAGTCAGT 200 GGAGCGCTCT GACGCCGGCC GGTACTGGTG CCAGGTGGAG GATGGGGGTG250 AAACCGAGAT CTCCCAGCCA GTGTGGCTCA CGGTAGAAGG TGTGCCATTT 300TTCACAGTGG AGCCAAAAGA TCTGGCAGTG CCACCCAATG CCCCTTTCCA 350 ACTGTCTTGTGAGGCTGTGG GTCCCCCTGA ACCTGTTACC ATTGTCTGGT 400 GGAGAGGAAC TACGAAGATCGGGGGACCCG CTCCCTCTCC ATCTGTTTTA 450 AATGTAACAG GGGTGACCCA GAGCACCATGTTTTCCTGTG AAGCTCACAA 500 CCTAAAAGGC CTGGCCTCTT CTCGCACAGC CACTGTTCACCTTCAAGCAC 550 TGCCTGCAGC CCCCTTCAAC ATCACCGTGA CAAAGCTTTC CAGCAGCAAC600 GCTAGTGTGG CCTGGATGCC AGGTGCTGAT GGCCGAGCTC TGCTACAGTC 650CTGTACAGTT CAGGTGACAC AGGCCCCAGG AGGCTGGGAA GTCCTGGCTG 700 TTGTGGTCCCTGTGCCCCCC TTTACCTGCC TGCTCCGGGA CCTGGTGCCT 750 GCCACCAACT ACAGCCTCAGGGTGCGCTGT GCCAATGCCT TGGGGCCCTC 800 TCCCTATGCT GACTGGGTGC CCTTTCAGACCAAGGGTCTA GCCCCAGCCA 850 GCGCTCCCCA AAACCTCCAT GCCATCCGCA CAGATTCAGGCCTCATCTTG 900 GAGTGGGAAG AAGTGATCCC CGAGGCCCCT TTGGAAGGCC CCCTGGGACC950 CTACAAACTG TCCTGGGTTC AAGACAATGG AACCCAGGAT GAGCTGACAG 1000TGGAGGGGAC CAGGGCCAAT TTGACAGGCT GGGATCCCCA AAAGGACCTG 1050 ATCGTACGTGTGTGCGTCTC CAATGCAGTT GGCTGTGGAC CCTGGAGTCA 1100 GCCACTGGTG GTCTCTTCTCATGACCGTGC AGGCCAGCAG GGCCCTCCTC 1150 ACAGCCGCAC ATCC 1164 388 aminoacids amino acid linear 6 Ala Gly Leu Lys Leu Met Gly Ala Pro Val LysLeu Thr Val Ser 1 5 10 15 Gln Gly Gln Pro Val Lys Leu Asn Cys Ser ValGlu Gly Met Glu 20 25 30 Glu Pro Asp Ile Gln Trp Val Lys Asp Gly Ala ValVal Gln Asn 35 40 45 Leu Asp Gln Leu Tyr Ile Pro Val Ser Glu Gln His TrpIle Gly 50 55 60 Phe Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala Gly ArgTyr 65 70 75 Trp Cys Gln Val Glu Asp Gly Gly Glu Thr Glu Ile Ser Gln Pro80 85 90 Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro 95100 105 Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 110115 120 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Val Trp Trp Arg 125130 135 Gly Thr Thr Lys Ile Gly Gly Pro Ala Pro Ser Pro Ser Val Leu 140145 150 Asn Val Thr Gly Val Thr Gln Ser Thr Met Phe Ser Cys Glu Ala 155160 165 His Asn Leu Lys Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His 170175 180 Leu Gln Ala Leu Pro Ala Ala Pro Phe Asn Ile Thr Val Thr Lys 185190 195 Leu Ser Ser Ser Asn Ala Ser Val Ala Trp Met Pro Gly Ala Asp 200205 210 Gly Arg Ala Leu Leu Gln Ser Cys Thr Val Gln Val Thr Gln Ala 215220 225 Pro Gly Gly Trp Glu Val Leu Ala Val Val Val Pro Val Pro Pro 230235 240 Phe Thr Cys Leu Leu Arg Asp Leu Val Pro Ala Thr Asn Tyr Ser 245250 255 Leu Arg Val Arg Cys Ala Asn Ala Leu Gly Pro Ser Pro Tyr Ala 260265 270 Asp Trp Val Pro Phe Gln Thr Lys Gly Leu Ala Pro Ala Ser Ala 275280 285 Pro Gln Asn Leu His Ala Ile Arg Thr Asp Ser Gly Leu Ile Leu 290295 300 Glu Trp Glu Glu Val Ile Pro Glu Ala Pro Leu Glu Gly Pro Leu 305310 315 Gly Pro Tyr Lys Leu Ser Trp Val Gln Asp Asn Gly Thr Gln Asp 320325 330 Glu Leu Thr Val Glu Gly Thr Arg Ala Asn Leu Thr Gly Trp Asp 335340 345 Pro Gln Lys Asp Leu Ile Val Arg Val Cys Val Ser Asn Ala Val 350355 360 Gly Cys Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser His Asp 365370 375 Arg Ala Gly Gln Gln Gly Pro Pro His Ser Arg Thr Ser 380 385 3881197 bases nucleic acid single linear 7 GATGCTGACA TGAAGGGACA TTTTGATCCTGCCAAGTGCC GCTATGCCCT 50 GGGCATGCAG GACCGGACCA TCCCAGACAG TGACATCTCTGCTTCCAGCT 100 CCTGGTCAGA TTCCACTGCC GCCCGCCACA GCAGGTTGGA GAGCAGTGAC150 GGGGATGGGG CCTGGTGCCC CGCAGGGTCG GTGTTTCCCA AGGAGGAGGA 200GTACTTGCAG GTGGATCTAC AACGACTGCA CCTGGTGGCT CTGGTGGGCA 250 CCCAGGGACGGCATGCCGGG GGCCTGGGCA AGGAGTTCTC CCGGAGCTAC 300 CGGCTGCGTT ACTCCCGGGATGGTCGCCGC TGGATGGGCT GGAAGGACCG 350 CTGGGGTCAG GAGGTGATCT CAGGCAATGAGGACCCTGAG GGAGTGGTGC 400 TGAAGGACCT TGGGCCCCCC ATGGTTGCCC GACTGGTTCGCTTCTACCCC 450 CGGGCTGACC GGGTCATGAG CGTCTGTCTG CGGGTAGAGC TCTATGGCTG500 CCTCTGGAGG GATGGACTCC TGTCTTACAC CGCCCCTGTG GGGCAGACAA 550TGTATTTATC TGAGGCCGTG TACCTCAACG ACTCCACCTA TGACGGACAT 600 ACCGTGGGCGGACTGCAGTA TGGGGGTCTG GGCCAGCTGG CAGATGGTGT 650 GGTGGGGCTG GATGACTTTAGGAAGAGTCA GGAGCTGCGG GTCTGGCCAG 700 GCTATGACTA TGTGGGATGG AGCAACCACAGCTTCTCCAG TGGCTATGTG 750 GAGATGGAGT TTGAGTTTGA CCGGCTGAGG GCCTTCCAGGCTATGCAGGT 800 CCACTGTAAC AACATGCACA CGCTGGGAGC CCGTCTGCCT GGCGGGGTGG850 AATGTCGCTT CCGGCGTGGC CCTGCCATGG CCTGGGAGGG GGAGCCCATG 900CGCCACAACC TAGGGGGCAA CCTGGGGGAC CCCAGAGCCC GGGCTGTCTC 950 AGTGCCCCTTGGCGGCCGTG TGGCTCGCTT TCTGCAGTGC CGCTTCCTCT 1000 TTGCGGGGCC CTGGTTACTCTTCAGCGAAA TCTCCTTCAT CTCTGATGTG 1050 GTGAACAATT CCTCTCCGGC ACTGGGAGGCACCTTCCCGC CAGCCCCCTG 1100 GTGGCCGCCT GGCCCACCTC CCACCAACTT CAGCAGCTTGGAGCTGGAGC 1150 CCAGAGGCCA GCAGCCCGTG GCCAAGCCCG AGGGGAGCCC GACCGCC 1197399 amino acids amino acid linear 8 Asp Ala Asp Met Lys Gly His Phe AspPro Ala Lys Cys Arg Tyr 1 5 10 15 Ala Leu Gly Met Gln Asp Arg Thr IlePro Asp Ser Asp Ile Ser 20 25 30 Ala Ser Ser Ser Trp Ser Asp Ser Thr AlaAla Arg His Ser Arg 35 40 45 Leu Glu Ser Ser Asp Gly Asp Gly Ala Trp CysPro Ala Gly Ser 50 55 60 Val Phe Pro Lys Glu Glu Glu Tyr Leu Gln Val AspLeu Gln Arg 65 70 75 Leu His Leu Val Ala Leu Val Gly Thr Gln Gly Arg HisAla Gly 80 85 90 Gly Leu Gly Lys Glu Phe Ser Arg Ser Tyr Arg Leu Arg TyrSer 95 100 105 Arg Asp Gly Arg Arg Trp Met Gly Trp Lys Asp Arg Trp GlyGln 110 115 120 Glu Val Ile Ser Gly Asn Glu Asp Pro Glu Gly Val Val LeuLys 125 130 135 Asp Leu Gly Pro Pro Met Val Ala Arg Leu Val Arg Phe TyrPro 140 145 150 Arg Ala Asp Arg Val Met Ser Val Cys Leu Arg Val Glu LeuTyr 155 160 165 Gly Cys Leu Trp Arg Asp Gly Leu Leu Ser Tyr Thr Ala ProVal 170 175 180 Gly Gln Thr Met Tyr Leu Ser Glu Ala Val Tyr Leu Asn AspSer 185 190 195 Thr Tyr Asp Gly His Thr Val Gly Gly Leu Gln Tyr Gly GlyLeu 200 205 210 Gly Gln Leu Ala Asp Gly Val Val Gly Leu Asp Asp Phe ArgLys 215 220 225 Ser Gln Glu Leu Arg Val Trp Pro Gly Tyr Asp Tyr Val GlyTrp 230 235 240 Ser Asn His Ser Phe Ser Ser Gly Tyr Val Glu Met Glu PheGlu 245 250 255 Phe Asp Arg Leu Arg Ala Phe Gln Ala Met Gln Val His CysAsn 260 265 270 Asn Met His Thr Leu Gly Ala Arg Leu Pro Gly Gly Val GluCys 275 280 285 Arg Phe Arg Arg Gly Pro Ala Met Ala Trp Glu Gly Glu ProMet 290 295 300 Arg His Asn Leu Gly Gly Asn Leu Gly Asp Pro Arg Ala ArgAla 305 310 315 Val Ser Val Pro Leu Gly Gly Arg Val Ala Arg Phe Leu GlnCys 320 325 330 Arg Phe Leu Phe Ala Gly Pro Trp Leu Leu Phe Ser Glu IleSer 335 340 345 Phe Ile Ser Asp Val Val Asn Asn Ser Ser Pro Ala Leu GlyGly 350 355 360 Thr Phe Pro Pro Ala Pro Trp Trp Pro Pro Gly Pro Pro ProThr 365 370 375 Asn Phe Ser Ser Leu Glu Leu Glu Pro Arg Gly Gln Gln ProVal 380 385 390 Ala Lys Pro Glu Gly Ser Pro Thr Ala 395 399 3785 basesnucleic acid single linear 9 CCTCCGCCAC CCTCCTCTCA GCGCTCGCGG GCCGGGCCCGGCATGGTGCG 50 CGTCGCCGCC GATGGCGCTG AGGCGGAGCA TGGGGTGGCC GGGGCTCCGG 100CCGCTGCTGC TGGCGGGACT GGCTTCTCTG CTGCTCCCCG GGTCTGCGGC 150 CGCAGGCCTGAAGCTCATGG GCGCCCCAGT GAAGATGACC GTGTCTCAGG 200 GGCAGCCAGT GAAGCTCAACTGCAGCGTGG AGGGGATGGA GGACCCTGAC 250 ATCCACTGGA TGAAGGATGG CACCGTGGTCCAGAATGCAA GCCAGGTGTC 300 CATCTCCATC AGCGAGCACA GCTGGATTGG CTTACTCAGCCTAAAGTCAG 350 TGGAGCGGTC TGATGCTGGC CTGTACTGGT GCCAGGTGAA GGATGGGGAG400 GAAACCAAGA TCTCTCAGTC AGTATGGCTC ACTGTCGAAG GTGTGCCATT 450CTTCACAGTG GAACCAAAAG ATCTGGCGGT GCCACCCAAT GCCCCTTTTC 500 AGCTGTCTTGTGAGGCTGTG GGTCCTCCAG AACCCGTAAC CATTTACTGG 550 TGGAGAGGAC TCACTAAGGTTGGGGGACCT GCTCCCTCTC CCTCTGTTTT 600 AAATGTGACA GGAGTGACCC AGCGCACAGAGTTTTCTTGT GAAGCCCGCA 650 ACATAAAAGG CCTGGCCACT TCCCGACCAG CCATTGTTCGCCTTCAAGCA 700 CCGCCTGCAG CTCCTTTCAA CACCACAGTA ACAACGATCT CCAGCTACAA750 CGCTAGCGTG GCCTGGGTGC CAGGTGCTGA CGGCCTAGCT CTGCTGCATT 800CCTGTACTGT ACAGGTGGCA CACGCCCCAG GAGAATGGGA GGCCCTTGCT 850 GTTGTGGTTCCTGTGCCACC TTTTACCTGC CTGCTTCGGA ACTTGGCCCC 900 TGCCACCAAC TACAGCCTTAGGGTGCGCTG TGCCAATGCC TTGGGCCCTT 950 CTCCCTACGG CGACTGGGTG CCCTTTCAGACAAAGGGCCT AGCGCCAGCC 1000 AGAGCTCCTC AGAATTTCCA TGCCATTCGT ACCGACTCAGGCCTTATCCT 1050 GGAATGGGAA GAAGTGATTC CTGAAGACCC TGGGGAAGGC CCCCTAGGAC1100 CTTATAAGCT GTCCTGGGTC CAAGAAAATG GAACCCAGGA TGAGCTGATG 1150GTGGAAGGGA CCAGGGCCAA TCTGACCGAC TGGGATCCCC AGAAGGACCT 1200 GATTTTGCGTGTGTGTGCCT CCAATGCAAT TGGTGATGGG CCCTGGAGTC 1250 AGCCACTGGT GGTGTCTTCTCATGACCATG CAGGGAGGCA GGGCCCTCCC 1300 CACAGCCGCA CATCCTGGGT GCCTGTGGTCCTGGGCGTGC TCACCGCCCT 1350 GATCACAGCT GCTGCCTTGG CCCTCATCCT GCTTCGGAAGAGACGCAAGG 1400 AGACGCGTTT CGGGCAAGCC TTTGACAGTG TCATGGCCCG AGGGGAGCCA1450 GCTGTACACT TCCGGGCAGC CCGATCTTTC AATCGAGAAA GGCCTGAACG 1500CATTGAGGCC ACATTGGATA GCCTGGGCAT CAGCGATGAA TTGAAGGAAA 1550 AGCTGGAGGATGTCCTCATT CCAGAGCAGC AGTTCACCCT CGGTCGGATG 1600 TTGGGCAAAG GAGAGTTTGGATCAGTGCGG GAAGCCCAGC TAAAGCAGGA 1650 AGATGGCTCC TTCGTGAAAG TGGCAGTGAAGATGCTGAAA GCTGACATCA 1700 TTGCCTCAAG CGACATAGAA GAGTTCCTCC GGGAAGCAGCTTGCATGAAG 1750 GAGTTTGACC ATCCACACGT GGCCAAGCTT GTTGGGGTGA GCCTCCGGAG1800 CAGGGCTAAA GGTCGTCTCC CCATTCCCAT GGTCATCCTG CCCTTCATGA 1850AACATGGAGA CTTGCACGCC TTTCTGCTCG CCTCCCGAAT CGGGGAGAAC 1900 CCTTTTAACCTGCCCCTGCA GACCCTGGTC CGGTTCATGG TGGACATTGC 1950 CTGTGGCATG GAGTACCTGAGCTCCCGGAA CTTCATCCAC CGAGACCTAG 2000 CAGCTCGGAA TTGCATGCTG GCCGAGGACATGACAGTGTG TGTGGCTGAT 2050 TTTGGACTCT CTCGGAAAAT CTATAGCGGG GACTATTATCGTCAGGGCTG 2100 TGCCTCCAAA TTGCCCGTCA AGTGGCTGGC CCTGGAGAGC TTGGCTGACA2150 ACTTGTATAC TGTACACAGT GATGTGTGGG CCTTCGGGGT GACCATGTGG 2200GAGATCATGA CTCGTGGGCA GACGCCATAT GCTGGCATTG AAAATGCTGA 2250 GATTTACAACTACCTCATCG GCGGGAACCG CCTGAAGCAG CCTCCGGAGT 2300 GCATGGAGGA AGTGTATGATCTCATGTACC AGTGCTGGAG CGCCGACCCC 2350 AAGCAGCGCC CAAGCTTCAC GTGTCTGCGAATGGAACTGG AGAACATTCT 2400 GGGCCACCTG TCTGTGCTGT CCACCAGCCA GGACCCCTTGTACATCAACA 2450 TTGAGAGAGC TGAGCAGCCT ACTGAGAGTG GCAGCCCTGA GCTGCACTGT2500 GGAGAGCGAT CCAGCAGCGA GGCAGGGGAC GGCAGTGGCG TGGGGGCAGT 2550AGGTGGCATC CCCAGTGACT CTCGGTACAT CTTCAGCCCC GGAGGGCTAT 2600 CCGAGTCACCAGGGCAGCTG GAGCAGCAGC CAGAAAGCCC CCTCAATGAG 2650 AACCAGAGGC TGTTGTTGCTGCAGCAAGGG CTACTGCCTC ACAGTAGCTG 2700 TTAACCCTCA GGCAGAGGAA AGTTGGGGCCCCTGGCTCTG CTGACCGCTG 2750 CGCTGCCTGA CTAGGCCCAG TCTGATCACA GCCCAGGCAGCAAGGTATGG 2800 AGGCTCCTGT GGTAGCCCTC CCAAGCTGTG TGGCGCCTGG ACGGACCAAA2850 TTGCCCAATC CCAGTTCTTC CTGCAGCCGC TCTGGCCAGC CTGGCATCAG 2900TTCAGGCCTT GGCTTAGAGG AGGTGAGCCA GAGCTGGTTG CCTGAATGCA 2950 GGCAGCTGGCAGGAGGGGAG GGTGGCTATG TTTCCATGGG TACCATGGGT 3000 GTGGATGGCA GTAAGGGAGGGTAGCAACAG CCTGTGGGCC CCTACCCTCC 3050 TGGCTGAGCT GCTCCTACTT TAGTGCATGCTTGGAGCCGC CTGCAGCCTG 3100 GAACTCAGCA CTGCCCACCA CACTTGGGCC GAAATGCCAGGTTTGCCCCT 3150 CTTAAGTCAC AAAGAGATGT CCATGTATTG TTCCCTTTTA GGTGATGATT3200 AGGAAGGGAT TGGCACACTT GGGTCCCTAA GCCCTATGGC AGGAAATGGT 3250GGGATATTCT CAGGTCTGAA TCCTCATCAT CTTCCTGATT CCCCACCCTG 3300 CAAAGGCCTGGAACTGGCTG TGGGGCTCTG ACGCATGCTG AAGGACAAAA 3350 GGTTACAGAG ATCCGACTTCAAAAGGCAGG GTCTGAGTCT GGCAGGTGGA 3400 GAGGTGCTAA GGGGCTGGCC CAGGAGTCAGGCATTTCAGG ACCCCTCCAA 3450 GCTTCTACAG TCTGTCTGAG CATGCTACCA AGCCCCCAGATACCCCAAAA 3500 CTAACAGAGG CAGTTTTGTC TGAGCCCAGC CCTCCCACAT GATGACCCTT3550 AGGTCTACCC TCCTCTCTAA ATGGACATCC TCGTTTGTCC CAAGTCTCCA 3600GAGAGACTAC TGATGGCTGA TGTGGGTAAG AAAAGTTCCA GGAACCAGGG 3650 CTGGGGTGGAACCAGGGCTG GGGTCGAGGC AGGCTCTTGG GCAGGCTCTT 3700 GCTGTTAGGA ACATTTCTAAGCTATTAAGT TGCTGTTTCA AAACAAATAA 3750 AATTGAAACA TAAAGAATCA AAAAAAAAAAAAAAA 3785 880 amino acids amino acid linear 10 Met Ala Leu Arg Arg SerMet Gly Trp Pro Gly Leu Arg Pro Leu 1 5 10 15 Leu Leu Ala Gly Leu AlaSer Leu Leu Leu Pro Gly Ser Ala Ala 20 25 30 Ala Gly Leu Lys Leu Met GlyAla Pro Val Lys Met Thr Val Ser 35 40 45 Gln Gly Gln Pro Val Lys Leu AsnCys Ser Val Glu Gly Met Glu 50 55 60 Asp Pro Asp Ile His Trp Met Lys AspGly Thr Val Val Gln Asn 65 70 75 Ala Ser Gln Val Ser Ile Ser Ile Ser GluHis Ser Trp Ile Gly 80 85 90 Leu Leu Ser Leu Lys Ser Val Glu Arg Ser AspAla Gly Leu Tyr 95 100 105 Trp Cys Gln Val Lys Asp Gly Glu Glu Thr LysIle Ser Gln Ser 110 115 120 Val Trp Leu Thr Val Glu Gly Val Pro Phe PheThr Val Glu Pro 125 130 135 Lys Asp Leu Ala Val Pro Pro Asn Ala Pro PheGln Leu Ser Cys 140 145 150 Glu Ala Val Gly Pro Pro Glu Pro Val Thr IleTyr Trp Trp Arg 155 160 165 Gly Leu Thr Lys Val Gly Gly Pro Ala Pro SerPro Ser Val Leu 170 175 180 Asn Val Thr Gly Val Thr Gln Arg Thr Glu PheSer Cys Glu Ala 185 190 195 Arg Asn Ile Lys Gly Leu Ala Thr Ser Arg ProAla Ile Val Arg 200 205 210 Leu Gln Ala Pro Pro Ala Ala Pro Phe Asn ThrThr Val Thr Thr 215 220 225 Ile Ser Ser Tyr Asn Ala Ser Val Ala Trp ValPro Gly Ala Asp 230 235 240 Gly Leu Ala Leu Leu His Ser Cys Thr Val GlnVal Ala His Ala 245 250 255 Pro Gly Glu Trp Glu Ala Leu Ala Val Val ValPro Val Pro Pro 260 265 270 Phe Thr Cys Leu Leu Arg Asn Leu Ala Pro AlaThr Asn Tyr Ser 275 280 285 Leu Arg Val Arg Cys Ala Asn Ala Leu Gly ProSer Pro Tyr Gly 290 295 300 Asp Trp Val Pro Phe Gln Thr Lys Gly Leu AlaPro Ala Arg Ala 305 310 315 Pro Gln Asn Phe His Ala Ile Arg Thr Asp SerGly Leu Ile Leu 320 325 330 Glu Trp Glu Glu Val Ile Pro Glu Asp Pro GlyGlu Gly Pro Leu 335 340 345 Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu AsnGly Thr Gln Asp 350 355 360 Glu Leu Met Val Glu Gly Thr Arg Ala Asn LeuThr Asp Trp Asp 365 370 375 Pro Gln Lys Asp Leu Ile Leu Arg Val Cys AlaSer Asn Ala Ile 380 385 390 Gly Asp Gly Pro Trp Ser Gln Pro Leu Val ValSer Ser His Asp 395 400 405 His Ala Gly Arg Gln Gly Pro Pro His Ser ArgThr Ser Trp Val 410 415 420 Pro Val Val Leu Gly Val Leu Thr Ala Leu IleThr Ala Ala Ala 425 430 435 Leu Ala Leu Ile Leu Leu Arg Lys Arg Arg LysGlu Thr Arg Phe 440 445 450 Gly Gln Ala Phe Asp Ser Val Met Ala Arg GlyGlu Pro Ala Val 455 460 465 His Phe Arg Ala Ala Arg Ser Phe Asn Arg GluArg Pro Glu Arg 470 475 480 Ile Glu Ala Thr Leu Asp Ser Leu Gly Ile SerAsp Glu Leu Lys 485 490 495 Glu Lys Leu Glu Asp Val Leu Ile Pro Glu GlnGln Phe Thr Leu 500 505 510 Gly Arg Met Leu Gly Lys Gly Glu Phe Gly SerVal Arg Glu Ala 515 520 525 Gln Leu Lys Gln Glu Asp Gly Ser Phe Val LysVal Ala Val Lys 530 535 540 Met Leu Lys Ala Asp Ile Ile Ala Ser Ser AspIle Glu Glu Phe 545 550 555 Leu Arg Glu Ala Ala Cys Met Lys Glu Phe AspHis Pro His Val 560 565 570 Ala Lys Leu Val Gly Val Ser Leu Arg Ser ArgAla Lys Gly Arg 575 580 585 Leu Pro Ile Pro Met Val Ile Leu Pro Phe MetLys His Gly Asp 590 595 600 Leu His Ala Phe Leu Leu Ala Ser Arg Ile GlyGlu Asn Pro Phe 605 610 615 Asn Leu Pro Leu Gln Thr Leu Val Arg Phe MetVal Asp Ile Ala 620 625 630 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn PheIle His Arg Asp 635 640 645 Leu Ala Ala Arg Asn Cys Met Leu Ala Glu AspMet Thr Val Cys 650 655 660 Val Ala Asp Phe Gly Leu Ser Arg Lys Ile TyrSer Gly Asp Tyr 665 670 675 Tyr Arg Gln Gly Cys Ala Ser Lys Leu Pro ValLys Trp Leu Ala 680 685 690 Leu Glu Ser Leu Ala Asp Asn Leu Tyr Thr ValHis Ser Asp Val 695 700 705 Trp Ala Phe Gly Val Thr Met Trp Glu Ile MetThr Arg Gly Gln 710 715 720 Thr Pro Tyr Ala Gly Ile Glu Asn Ala Glu IleTyr Asn Tyr Leu 725 730 735 Ile Gly Gly Asn Arg Leu Lys Gln Pro Pro GluCys Met Glu Glu 740 745 750 Val Tyr Asp Leu Met Tyr Gln Cys Trp Ser AlaAsp Pro Lys Gln 755 760 765 Arg Pro Ser Phe Thr Cys Leu Arg Met Glu LeuGlu Asn Ile Leu 770 775 780 Gly His Leu Ser Val Leu Ser Thr Ser Gln AspPro Leu Tyr Ile 785 790 795 Asn Ile Glu Arg Ala Glu Gln Pro Thr Glu SerGly Ser Pro Glu 800 805 810 Leu His Cys Gly Glu Arg Ser Ser Ser Glu AlaGly Asp Gly Ser 815 820 825 Gly Val Gly Ala Val Gly Gly Ile Pro Ser AspSer Arg Tyr Ile 830 835 840 Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro GlyGln Leu Glu Gln 845 850 855 Gln Pro Glu Ser Pro Leu Asn Glu Asn Gln ArgLeu Leu Leu Leu 860 865 870 Gln Gln Gly Leu Leu Pro His Ser Ser Cys 875880 1164 bases nucleic acid single linear 11 GCAGGCCTGA AGCTCATGGGCGCCCCAGTG AAGATGACCG TGTCTCAGGG 50 GCAGCCAGTG AAGCTCAACT GCAGCGTGGAGGGGATGGAG GACCCTGACA 100 TCCACTGGAT GAAGGATGGC ACCGTGGTCC AGAATGCAAGCCAGGTGTCC 150 ATCTCCATCA GCGAGCACAG CTGGATTGGC TTACTCAGCC TAAAGTCAGT200 GGAGCGGTCT GATGCTGGCC TGTACTGGTG CCAGGTGAAG GATGGGGAGG 250AAACCAAGAT CTCTCAGTCA GTATGGCTCA CTGTCGAAGG TGTGCCATTC 300 TTCACAGTGGAACCAAAAGA TCTGGCGGTG CCACCCAATG CCCCTTTTCA 350 GCTGTCTTGT GAGGCTGTGGGTCCTCCAGA ACCCGTAACC ATTTACTGGT 400 GGAGAGGACT CACTAAGGTT GGGGGACCTGCTCCCTCTCC CTCTGTTTTA 450 AATGTGACAG GAGTGACCCA GCGCACAGAG TTTTCTTGTGAAGCCCGCAA 500 CATAAAAGGC CTGGCCACTT CCCGACCAGC CATTGTTCGC CTTCAAGCAC550 CGCCTGCAGC TCCTTTCAAC ACCACAGTAA CAACGATCTC CAGCTACAAC 600GCTAGCGTGG CCTGGGTGCC AGGTGCTGAC GGCCTAGCTC TGCTGCATTC 650 CTGTACTGTACAGGTGGCAC ACGCCCCAGG AGAATGGGAG GCCCTTGCTG 700 TTGTGGTTCC TGTGCCACCTTTTACCTGCC TGCTTCGGAA CTTGGCCCCT 750 GCCACCAACT ACAGCCTTAG GGTGCGCTGTGCCAATGCCT TGGGCCCTTC 800 TCCCTACGGC GACTGGGTGC CCTTTCAGAC AAAGGGCCTAGCGCCAGCCA 850 GAGCTCCTCA GAATTTCCAT GCCATTCGTA CCGACTCAGG CCTTATCCTG900 GAATGGGAAG AAGTGATTCC TGAAGACCCT GGGGAAGGCC CCCTAGGACC 950TTATAAGCTG TCCTGGGTCC AAGAAAATGG AACCCAGGAT GAGCTGATGG 1000 TGGAAGGGACCAGGGCCAAT CTGACCGACT GGGATCCCCA GAAGGACCTG 1050 ATTTTGCGTG TGTGTGCCTCCAATGCAATT GGTGATGGGC CCTGGAGTCA 1100 GCCACTGGTG GTGTCTTCTC ATGACCATGCAGGGAGGCAG GGCCCTCCCC 1150 ACAGCCGCAC ATCC 1164 388 amino acids aminoacid linear 12 Ala Gly Leu Lys Leu Met Gly Ala Pro Val Lys Met Thr ValSer 1 5 10 15 Gln Gly Gln Pro Val Lys Leu Asn Cys Ser Val Glu Gly MetGlu 20 25 30 Asp Pro Asp Ile His Trp Met Lys Asp Gly Thr Val Val Gln Asn35 40 45 Ala Ser Gln Val Ser Ile Ser Ile Ser Glu His Ser Trp Ile Gly 5055 60 Leu Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala Gly Leu Tyr 65 7075 Trp Cys Gln Val Lys Asp Gly Glu Glu Thr Lys Ile Ser Gln Ser 80 85 90Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu Pro 95 100 105Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 110 115 120Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Tyr Trp Trp Arg 125 130 135Gly Leu Thr Lys Val Gly Gly Pro Ala Pro Ser Pro Ser Val Leu 140 145 150Asn Val Thr Gly Val Thr Gln Arg Thr Glu Phe Ser Cys Glu Ala 155 160 165Arg Asn Ile Lys Gly Leu Ala Thr Ser Arg Pro Ala Ile Val Arg 170 175 180Leu Gln Ala Pro Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr 185 190 195Ile Ser Ser Tyr Asn Ala Ser Val Ala Trp Val Pro Gly Ala Asp 200 205 210Gly Leu Ala Leu Leu His Ser Cys Thr Val Gln Val Ala His Ala 215 220 225Pro Gly Glu Trp Glu Ala Leu Ala Val Val Val Pro Val Pro Pro 230 235 240Phe Thr Cys Leu Leu Arg Asn Leu Ala Pro Ala Thr Asn Tyr Ser 245 250 255Leu Arg Val Arg Cys Ala Asn Ala Leu Gly Pro Ser Pro Tyr Gly 260 265 270Asp Trp Val Pro Phe Gln Thr Lys Gly Leu Ala Pro Ala Arg Ala 275 280 285Pro Gln Asn Phe His Ala Ile Arg Thr Asp Ser Gly Leu Ile Leu 290 295 300Glu Trp Glu Glu Val Ile Pro Glu Asp Pro Gly Glu Gly Pro Leu 305 310 315Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu Asn Gly Thr Gln Asp 320 325 330Glu Leu Met Val Glu Gly Thr Arg Ala Asn Leu Thr Asp Trp Asp 335 340 345Pro Gln Lys Asp Leu Ile Leu Arg Val Cys Ala Ser Asn Ala Ile 350 355 360Gly Asp Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser His Asp 365 370 375His Ala Gly Arg Gln Gly Pro Pro His Ser Arg Thr Ser 380 385 388 23 basesnucleic acid single linear 13 CGGATCCACA CGATGCGACT CTT 23 31 basesnucleic acid single linear 14 GGAATTCCTC TCATGGAGCT AGTCCATCTC T 31 21bases nucleic acid single linear 15 CGGATCCATC CACAGAGATG T 21 25 basesnucleic acid single linear 16 GGAATTCCAA AGGACCAGCA CGATC 25 40 basesnucleic acid single linear 17 GACCGTGTGT GTGGCTGACT TTGGACTCTCCTGGAAGATC 40 40 bases nucleic acid single linear 18 GGCTGTGCCTCCAAATTGCC CGTCAAGTGG CTGGCCCTGG 40 50 bases nucleic acid single linear19 AGCCGGTGAA GCTGAACTGC AGTGTGGAGG GGATGGAGGA GCCTGACATC 50 50 basesnucleic acid single linear 20 TCCAGCTACA ACGCTAGCGT GGCCTGGGTGCCAGGTGCTG ACGGCCTAGC 50 9 amino acids amino acid linear 21 Ile His ArgAsp Leu Ala Ala Arg Asn 1 5 9 6 amino acids amino acid linear 22 Lys TrpIle Ala Ile Glu 1 5 6 8 amino acids amino acid linear 23 Lys Thr Trp ThrMet Ala Pro Glu 1 5 8 6 amino acids amino acid linear 24 Lys Trp Leu AlaLeu Glu 1 5 6 6 amino acids amino acid linear 25 Lys Trp Met Ala Leu Glu1 5 6 30 bases nucleic acid single linear 26 CAGCTGCTCG AGGCAGGTCTGAAGCTCATG 30 30 bases nucleic acid single linear 27 GCATGAATTCATGGCACACC TTCTACCGTG 30 20 bases nucleic acid single linear 28CACTGAGCTG GCTGACTAAG 20 20 bases nucleic acid single linear 29CCTGATAGGC TGGGTACTCC 20 18 bases nucleic acid single linear 30AAGCCCGGAC TGACCAAA 18 20 bases nucleic acid single linear 31 GTGCGGAATCAGAAAGATGG 20 18 bases nucleic acid single linear 32 TCAAGACAAT GGAACCCA18 36 bases nucleic acid single linear 33 CATGGAATTC GGTGACCGATGTGCGGCTGT GAGGAG 36 894 amino acids amino acid linear 34 Met Ala TrpArg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp 1 5 10 15 Cys Leu AlaLeu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr 20 25 30 Gln Ala Glu GluSer Pro Phe Val Gly Asn Pro Gly Asn Ile Thr 35 40 45 Gly Ala Arg Gly LeuThr Gly Thr Leu Arg Cys Gln Leu Gln Val 50 55 60 Gln Gly Glu Pro Pro GluVal His Trp Leu Arg Asp Gly Gln Ile 65 70 75 Leu Glu Leu Ala Asp Ser ThrGln Thr Gln Val Pro Leu Gly Glu 80 85 90 Asp Glu Gln Asp Asp Trp Ile ValVal Ser Gln Leu Arg Ile Thr 95 100 105 Ser Leu Gln Leu Ser Asp Thr GlyGln Tyr Gln Cys Leu Val Phe 110 115 120 Leu Gly His Gln Thr Phe Val SerGln Pro Gly Tyr Val Gly Leu 125 130 135 Glu Gly Leu Pro Tyr Phe Leu GluGlu Pro Glu Asp Arg Thr Val 140 145 150 Ala Ala Asn Thr Pro Phe Asn LeuSer Cys Gln Ala Gln Gly Pro 155 160 165 Pro Glu Pro Val Asp Leu Leu TrpLeu Gln Asp Ala Val Pro Leu 170 175 180 Ala Thr Ala Pro Gly His Gly ProGln Arg Ser Leu His Val Pro 185 190 195 Gly Leu Asn Lys Thr Ser Ser PheSer Cys Glu Ala His Asn Ala 200 205 210 Lys Gly Val Thr Thr Ser Arg ThrAla Thr Ile Thr Val Leu Pro 215 220 225 Gln Gln Pro Arg Asn Leu His LeuVal Ser Arg Gln Pro Thr Glu 230 235 240 Leu Glu Val Ala Trp Thr Pro GlyLeu Ser Gly Ile Tyr Pro Leu 245 250 255 Thr His Cys Thr Leu Gln Ala ValLeu Ser Asp Asp Gly Met Gly 260 265 270 Ile Gln Ala Gly Glu Pro Asp ProPro Glu Glu Pro Leu Thr Ser 275 280 285 Gln Ala Ser Val Pro Pro His GlnLeu Arg Leu Gly Ser Leu His 290 295 300 Pro His Thr Pro Tyr His Ile ArgVal Ala Cys Thr Ser Ser Gln 305 310 315 Gly Pro Ser Ser Trp Thr His TrpLeu Pro Val Glu Thr Pro Glu 320 325 330 Gly Val Pro Leu Gly Pro Pro GluAsn Ile Ser Ala Thr Arg Asn 335 340 345 Gly Ser Gln Ala Phe Val His TrpGln Glu Pro Arg Ala Pro Leu 350 355 360 Gln Gly Thr Leu Leu Gly Tyr ArgLeu Ala Tyr Gln Gly Gln Asp 365 370 375 Thr Pro Glu Val Leu Met Asp IleGly Leu Arg Gln Glu Val Thr 380 385 390 Leu Glu Leu Gln Gly Asp Gly SerVal Ser Asn Leu Thr Val Cys 395 400 405 Val Ala Ala Tyr Thr Ala Ala GlyAsp Gly Pro Trp Ser Leu Pro 410 415 420 Val Pro Leu Glu Ala Trp Arg ProGly Gln Ala Gln Pro Val His 425 430 435 Gln Leu Val Lys Glu Pro Ser ThrPro Ala Phe Ser Trp Pro Trp 440 445 450 Trp Tyr Val Leu Leu Gly Ala ValVal Ala Ala Ala Cys Val Leu 455 460 465 Ile Leu Ala Leu Phe Leu Val HisArg Arg Lys Lys Glu Thr Arg 470 475 480 Tyr Gly Glu Val Phe Glu Pro ThrVal Glu Arg Gly Glu Leu Val 485 490 495 Val Arg Tyr Arg Val Arg Lys SerTyr Ser Arg Arg Thr Thr Glu 500 505 510 Ala Thr Leu Asn Ser Leu Gly IleSer Glu Glu Leu Lys Glu Lys 515 520 525 Leu Arg Asp Val Met Val Asp ArgHis Lys Val Ala Leu Gly Lys 530 535 540 Thr Leu Gly Glu Gly Glu Phe GlyAla Val Met Glu Gly Gln Leu 545 550 555 Asn Gln Asp Asp Ser Ile Leu LysVal Ala Val Lys Thr Met Lys 560 565 570 Ile Ala Ile Cys Thr Arg Ser GluLeu Glu Asp Phe Leu Ser Glu 575 580 585 Ala Val Cys Met Lys Glu Phe AspHis Pro Asn Val Met Arg Leu 590 595 600 Ile Gly Val Cys Phe Gln Gly SerGlu Arg Glu Ser Phe Pro Ala 605 610 615 Pro Val Val Ile Leu Pro Phe MetLys His Gly Asp Leu His Ser 620 625 630 Phe Leu Leu Tyr Ser Arg Leu GlyAsp Gln Pro Val Tyr Leu Pro 635 640 645 Thr Gln Met Leu Val Lys Phe MetAla Asp Ile Ala Ser Gly Met 650 655 660 Glu Tyr Leu Ser Thr Lys Arg PheIle His Arg Asp Leu Ala Ala 665 670 675 Arg Asn Cys Met Leu Asn Glu AsnMet Ser Val Cys Val Ala Asp 680 685 690 Phe Gly Leu Ser Lys Lys Ile TyrAsn Gly Asp Tyr Tyr Arg Gln 695 700 705 Gly Arg Ile Ala Lys Met Pro ValLys Trp Ile Ala Ile Glu Ser 710 715 720 Leu Ala Asp Arg Val Tyr Thr SerLys Ser Asp Val Trp Ser Phe 725 730 735 Gly Val Thr Met Trp Glu Ile AlaThr Arg Gly Gln Thr Pro Tyr 740 745 750 Pro Gly Val Glu Asn Ser Glu IleTyr Asp Tyr Leu Arg Gln Gly 755 760 765 Asn Arg Leu Lys Gln Pro Ala AspCys Leu Asp Gly Leu Tyr Ala 770 775 780 Leu Met Ser Arg Cys Trp Glu LeuAsn Pro Gln Asp Arg Pro Ser 785 790 795 Phe Thr Glu Leu Arg Glu Asp LeuGlu Asn Thr Leu Lys Ala Leu 800 805 810 Pro Pro Ala Gln Glu Pro Asp GluIle Leu Tyr Val Asn Met Asp 815 820 825 Glu Gly Gly Gly Tyr Pro Glu ProPro Gly Ala Ala Gly Gly Ala 830 835 840 Asp Pro Pro Thr Gln Pro Asp ProLys Asp Ser Cys Ser Cys Leu 845 850 855 Thr Ala Ala Glu Val His Pro AlaGly Arg Tyr Val Leu Cys Pro 860 865 870 Ser Thr Thr Pro Ser Pro Ala GlnPro Ala Asp Arg Gly Ser Pro 875 880 885 Ala Ala Pro Gly Gln Glu Asp GlyAla 890 894 888 amino acids amino acid linear 35 Met Gly Arg Val Pro LeuAla Trp Trp Leu Ala Leu Cys Cys Trp 1 5 10 15 Gly Cys Ala Ala His LysAsp Thr Gln Thr Glu Ala Gly Ser Pro 20 25 30 Phe Val Gly Asn Pro Gly AsnIle Thr Gly Ala Arg Gly Leu Thr 35 40 45 Gly Thr Leu Arg Cys Glu Leu GlnVal Gln Gly Glu Pro Pro Glu 50 55 60 Val Val Trp Leu Arg Asp Gly Gln IleLeu Glu Leu Ala Asp Asn 65 70 75 Thr Gln Thr Gln Val Pro Leu Gly Glu AspTrp Gln Asp Glu Trp 80 85 90 Lys Val Val Ser Gln Leu Arg Ile Ser Ala LeuGln Leu Ser Asp 95 100 105 Ala Gly Glu Tyr Gln Cys Met Val His Leu GluGly Arg Thr Phe 110 115 120 Val Ser Gln Pro Gly Phe Val Gly Leu Glu GlyLeu Pro Tyr Phe 125 130 135 Leu Glu Glu Pro Glu Asp Lys Ala Val Pro AlaAsn Thr Pro Phe 140 145 150 Asn Leu Ser Cys Gln Ala Gln Gly Pro Pro GluPro Val Thr Leu 155 160 165 Leu Trp Leu Gln Asp Ala Val Pro Leu Ala ProVal Thr Gly His 170 175 180 Ser Ser Gln His Ser Leu Gln Thr Pro Gly LeuAsn Lys Thr Ser 185 190 195 Ser Phe Ser Cys Glu Ala His Asn Ala Lys GlyVal Thr Thr Ser 200 205 210 Arg Thr Ala Thr Ile Thr Val Leu Pro Gln ArgPro His His Leu 215 220 225 His Val Val Ser Arg Gln Pro Thr Glu Leu GluVal Ala Trp Thr 230 235 240 Pro Gly Leu Ser Gly Ile Tyr Pro Leu Thr HisCys Asn Leu Gln 245 250 255 Ala Val Leu Ser Asp Asp Gly Val Gly Ile TrpLeu Gly Lys Ser 260 265 270 Asp Pro Pro Glu Asp Pro Leu Thr Leu Gln ValSer Val Pro Pro 275 280 285 His Gln Leu Arg Leu Glu Lys Leu Leu Pro HisThr Pro Tyr His 290 295 300 Ile Arg Ile Ser Cys Ser Ser Ser Gln Gly ProSer Pro Trp Thr 305 310 315 His Trp Leu Pro Val Glu Thr Thr Glu Gly ValPro Leu Gly Pro 320 325 330 Pro Glu Asn Val Ser Ala Met Arg Asn Gly SerGln Val Leu Val 335 340 345 Arg Trp Gln Glu Pro Arg Val Pro Leu Gln GlyThr Leu Leu Gly 350 355 360 Tyr Arg Leu Ala Tyr Arg Gly Gln Asp Thr ProGlu Val Leu Met 365 370 375 Asp Ile Gly Leu Thr Arg Glu Val Thr Leu GluLeu Arg Gly Asp 380 385 390 Arg Pro Val Ala Asn Leu Thr Val Ser Val ThrAla Tyr Thr Ser 395 400 405 Ala Gly Asp Gly Pro Trp Ser Leu Pro Val ProLeu Glu Pro Trp 410 415 420 Arg Pro Gly Gln Gly Gln Pro Leu His His LeuVal Ser Glu Pro 425 430 435 Pro Pro Arg Ala Phe Ser Trp Pro Trp Trp TyrVal Leu Leu Gly 440 445 450 Ala Leu Val Ala Ala Ala Cys Val Leu Ile LeuAla Leu Phe Leu 455 460 465 Val His Arg Arg Lys Lys Glu Thr Arg Tyr GlyGlu Val Phe Glu 470 475 480 Pro Thr Val Glu Arg Gly Glu Leu Val Val ArgTyr Arg Val Arg 485 490 495 Lys Ser Tyr Ser Arg Arg Thr Thr Glu Ala ThrLeu Asn Ser Leu 500 505 510 Gly Ile Ser Glu Glu Leu Lys Glu Lys Leu ArgAsp Val Met Val 515 520 525 Asp Arg His Lys Val Ala Leu Gly Lys Thr LeuGly Glu Gly Glu 530 535 540 Phe Gly Ala Val Met Glu Gly Gln Leu Asn GlnAsp Asp Ser Ile 545 550 555 Leu Lys Val Ala Val Lys Thr Met Lys Ile AlaIle Cys Thr Arg 560 565 570 Ser Glu Leu Glu Asp Phe Leu Ser Glu Ala ValCys Met Lys Glu 575 580 585 Phe Asp His Pro Asn Val Met Arg Leu Ile GlyVal Cys Phe Gln 590 595 600 Gly Ser Asp Arg Glu Gly Phe Pro Glu Pro ValVal Ile Leu Pro 605 610 615 Phe Met Lys His Gly Asp Leu His Ser Phe LeuLeu Tyr Ser Arg 620 625 630 Leu Gly Asp Gln Pro Val Phe Leu Pro Thr GlnMet Leu Val Lys 635 640 645 Phe Met Ala Asp Ile Ala Ser Gly Met Glu TyrLeu Ser Thr Lys 650 655 660 Arg Phe Ile His Arg Asp Leu Ala Ala Arg AsnCys Met Leu Asn 665 670 675 Glu Asn Met Ser Val Cys Val Ala Asp Phe GlyLeu Ser Lys Lys 680 685 690 Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly ArgIle Ala Lys Met 695 700 705 Pro Val Lys Trp Ile Ala Ile Glu Ser Leu AlaAsp Arg Val Tyr 710 715 720 Thr Ser Lys Ser Asp Val Trp Ser Phe Gly ValThr Met Trp Glu 725 730 735 Ile Ala Thr Arg Gly Gln Thr Pro Tyr Pro GlyVal Glu Asn Ser 740 745 750 Glu Ile Tyr Asp Tyr Leu Arg Gln Gly Asn ArgLeu Lys Gln Pro 755 760 765 Val Asp Phe Leu Asp Gly Leu Tyr Ser Leu MetSer Arg Cys Trp 770 775 780 Glu Leu Asn Pro Arg Asp Arg Pro Ser Phe AlaGlu Leu Arg Glu 785 790 795 Asp Leu Glu Asn Thr Leu Lys Ala Leu Pro ProAla Gln Glu Pro 800 805 810 Asp Glu Ile Leu Tyr Val Asn Met Asp Glu GlyGly Ser His Leu 815 820 825 Glu Pro Arg Gly Ala Ala Gly Gly Ala Asp ProPro Thr Gln Pro 830 835 840 Asp Pro Lys Asp Ser Cys Ser Cys Leu Thr AlaAla Asp Val His 845 850 855 Ser Ala Gly Arg Tyr Val Leu Cys Pro Ser ThrAla Pro Gly Pro 860 865 870 Thr Leu Ser Ala Asp Arg Gly Cys Pro Ala ProPro Gly Gln Glu 875 880 885 Asp Gly Ala 888

1-25. (Canceled)
 26. An isolated nucleic acid molecule comprising anucleotide sequence having at least about 90% sequence identity to anucleotide sequence encoding an HPTK6 polypeptide comprising the aminoacid sequence shown in SEQ ID NO:4, or the complement of the nucleotidethereof.
 27. The isolated nucleic acid molecule of claim 26, comprisinga nucleotide sequence that encodes the amino acid sequence shown in SEQID NO:
 8. 28. The isolated nucleic acid molecule comprising the HPTK6nucleotide sequence shown in SEQ ID NO:7.
 29. The isolated nucleic acidmolecule comprising the HPTK6 nucleotide sequence shown in SEQ ID NO:3.30. An isolated nucleic acid molecule comprising an HPTK6 nucleotidesequence that hybridizes to the complement of a nucleic acid sequencethat encodes the amino acid sequence shown in SEQ ID NO:4, wherein thehybridization occurs under stringent hybridization and wash conditions.31. An isolated nucleic acid molecule comprising an HPTK6 nucleotidesequence that hybridizes to the complement of a nucleic acid sequencethat encodes the amino acid sequence shown in SEQ ID NO: 8, wherein thehybridization occurs under stringent hybridization and wash conditions.32. A vector comprising a nucleic acid molecule of claim
 26. 33. Thevector of claim 32, wherein said nucleic acid molecule is operablylinked to control sequences recognized by a host cell transformed withthe vector.
 34. A host cell comprising the vector of claim
 33. 35. Thehost cell of claim 34, wherein said cell is a CHO cell, a yeast cell orE. coli.
 36. A vector comprising a nucleic acid molecule of claim 27.37. A vector comprising a nucleic acid molecule of claim
 28. 38. Avector comprising a nucleic acid molecule of claim
 29. 39. A vectorcomprising a nucleic acid molecule of claim
 30. 40. A vector comprisinga nucleic acid molecule of claim 31.